WO2006110309A9 - Transgenic mice expressing a unique b cell population and methods of use - Google Patents

Abstract

The present invention includes transgenic animals, such as transgenic mice, that express a dominant-negative form of RAG-I during the transitional stage of B lymphocyte development, permitting initial antigen receptor gene rearrangement but blocking receptor editing and/or receptor revision that occurs during later periods in development. In one aspect the transgenic animal includes in its genome an exogenous polynucleotide includes a coding sequence encoding a catalytically defective RAG-I polypeptide, or an analog thereof. The present invention also provides methods of making and using such transgenic animals.

Description

TRANSGENIC MICE EXPRESSING A UNIQUE B CELL POPULATION

AND METHODS OF USE

CONTINUING APPLICATION DATA

This application claims the benefit of U.S. Provisional Application Serial No. 60/665,406, filed March 26, 2005, which is incorporated by reference herein.

BACKGROUND

B lymphocytes proceed through a series of developmental stages to acquire a functional, non-self reactive antigen receptor (the B cell receptor, or BCR). The antigen-binding portion of the BCR includes immunoglobulin heavy and light chain polypeptides. Both types of polypeptides contain an amino- terminal domain which directly contacts antigen and exhibits great sequence variability, and one or more constant domains. The exon encoding the variable domain, but not the constant domain(s), is assembled from arrays of component variable (V), diversity (D, heavy chain only), and joining (J) gene segments by site-specific DNA rearrangement. This rearrangement process, called V(D)J recombination, underlies the diversity of BCRs (as well as T cell receptors) and is evolutionarily conserved in all jawed vertebrates (Bassing et al., Cell, 109 Suppl:S45-S55 (2002), Litman et al., Annu Rev Immunol, 17:109-147 (1999)).

V(D)J recombination proceeds through two distinct phases in which DNA double strand breaks (DSBs) are first introduced by the RAG proteins, perhaps with assistance by high mobility group proteins (the cleavage phase), and then subsequently repaired via the non-homologous end joining (NHEJ) pathway (the joining phase) (Fugmann et al., Annu Rev Immunol, 18:495-527, 2000). In the cleavage phase, the RAG proteins catalyze DSBs at the ends of antigen receptor gene segments, directed by flanking recombination signal sequences (RSSs) containing a conserved heptamer and nonamer sequence element separated by spacer DNA either 12 or 23 bp in length. RAG-mediated cleavage generates two distinct DNA ends: blunt, 5'ρhosphorylated signal ends terminating at the heptamer and coding ends covalently sealed in DNA hairpin structure (Roth et al., Proc Natl Acad Sci, 90:10788-10792 (1993), Schlissel et al., Genes Dev, 7:2520-2535 (1993)). In the joining phase, the hairpinned coding ends are first resolved and rendered accessible to enzymes that remove nucleotides or add them (Komori et al., Science, 261 :1171-1175 (1993)). The most plausible candidate for mediating hairpin opening is the recently described Artemis protein (Moshous et al., Cell, 105:177-186 (2001)), in the association with DNA-PKcs (Ma et al., Cell, 108:781-794 (2002) (for discussion, see Schlissel, Cell, 109:1-4 (2002)). Finally, ubiquitous factors involved in NHEJ, including Ku70, Ku86, DNA- PKcs, XRCC4 and DNA Ligase IV (LiglV), are recruited to reorganize and seal the DNA ends (Bassing et al., Cell, 109 Suppl:S45-S55 (2002)). As a result, pairs of coding segments and their associated RSSs become transposed to form coding joints and signal joints, respectively. Signal joints typically contain two RSSs fused heptamer-to-heptamer, but coding joints are often imprecise, containing sequence deletions or insertions bearing palindromic repeats and/or nontemplated nucleotides (Tonegawa, Nature, 302:575-581 (1983)). Over the last decade, the biochemical mechanisms that underlie V(D)J recombination have become better understood (for reviews, see Fugmann et al., Annu Rev Immunol, 18:495-527 (2000), and Brandt and Roth, Curr Opin Immunol, 14:224-229 (2002)). Efforts to characterize the protein-DNA complexes that support the initiation of V(D)J recombination and identify amino acid residues in RAG-I and RAG-2 involved in catalysis and in mediating relevant protein-protein and protein-DNA interactions have refined RAG-I and RAG-2 structure-function relationships and clarified how the RAG proteins assemble a nucleoprotein complex capable of cleaving a 12/23 RSS pair (for review, see Swanson, Immunol Rev, 200:90-114 (2004)).

The developmental stages through which B (and T) cells mature are characterized by an ordered series of V(D)J recombination events and selection processes that, if successfully completed, give rise to mature, non-self-reactive lymphocytes (Wang and Clark, Immunology, 110:411-420 (2003)). Early in B cell development (which in an adult occurs in the bone marrow), the immunoglobulin heavy chain locus undergoes V(D)J rearrangement. The rearranged gene is functionally tested through the attempted pairing of the expressed μ heavy chain with the invariant polypeptides λ5 and Vpre-B (including the surrogate light chain, ψL). Successful pairing leads to the surface expression of the complex, called the pre-B cell receptor, heavy chain allelic exclusion, down-regulation of the V(D)J recombinase, closure of the heavy chain locus, and cell proliferation. The cells then exit cell cycle and enter a developmental stage during which the V(D)J recombinase is upregulated and light chain gene rearrangement ensues. Like the heavy chain gene, the rearranged light chain gene is functionally tested by pairing the expressed light chain with the μ heavy chain. Successful pairing leads to the expression of IgM on the cell surface (slgM), the phenotypic hallmark of an immature B cell. At this time, the cell begins to migrate out of the bone marrow, into the blood stream, and then to the spleen. As it does so, slgD begin to appear through alternative splicing of the heavy chain RNA transcript. During this transitional period, the BCR is tested for self-reactivity. Cells whose BCRs recognize self-antigen can undergo developmental arrest and reinitiate V(D)J recombination in order to "edit" receptor specificity away from autoreactivity (Jankovic et al., Annu Rev Immunol, 22:485-501 (2004)). Most often, this "receptor editing" process involves either the replacement of the offending light chain variable ex on or kappa deletion to promote λ light chain rearrangement (Gay et al., J. Exp Med, 177:999-1008 (1993), Tiegs et al., J Exp Med, 177:1009-1020 (1993), Retter and Nemazee, J Exp Med 188:1231-1238 (1998)). Rarely, heavy chain gene replacement may occur which involves recombination at a cryptic heptamer embedded in the 3' end of the rearranged V_H segment (Chen et al., Immunity, 3:747-755 (1995)). Receptor editing may therefore be considered a salvage pathway for potentially autoreactive B cells. The precise development stage at which receptor editing normally occurs remains unclear.

When the cell migrates out of the bone marrow, into the blood stream, and into the spleen, it referentially resides in the periarteriolar lymphoid sheath (PALS) surrounding a central arteriole. It is during this period of B cell development, called the transitional Tl stage, IgD begins to appear on the cell surface (slgD) through alternative splicing of the heavy chain RNA transcript. As the cell transitions into the next developmental stage, the T2 B cell, it migrates to the lymphoid follicle surrounding the PALS. Significant functional differences exist in the response of Tl and T2 B cells to antigenic stimulation. For example, Tl B cells fail to proliferate upon BCR cross-linking, which instead promotes apoptosis, whereas BCR cross-linking of T2 B cells causes proliferative expansion and induction of signals that promote cell survival and differentiation. Adoptive transfer experiments suggest that T2 cells can subsequently differentiate into follicular mature (FM) B cells. A third transitional B cell population, designated T3, has also been described, which may be distinguished from the other two based on IgM and IgD expression. The origin and fate of T3 B cells remain unclear, but there is recent evidence suggesting that T3 B cells and anergic B cells may be one in the same.

Two other minor B cell populations evident in spleen include marginal zone (MZ) B cells and CD5⁺ Bl (B-Ia) B cells. Unlike T2 and FM B cells, MZ B cells are localized to the marginal sinus, and express lower levels of CD23 and slgD. The MZ B cell is thought to originate from a T2 precursor, although evidence supporting its differentiation from the FM B cell has also been discussed. The anatomic location of MZ B cells allows them to quickly respond to blood-borne pathogens, especially those opsonized by complement, as MZ B cells express high levels of complement receptor 2 (CD21). They have the additional capability of rapidly maturing into plasmablasts after activation. Bl B cells, like MZ B cells, tend to occupy a specific niche in the host (Hardy and Hayakawa, Adv Immunol 55:297-339 (1994), Berland and Wortis, Annu Rev Immunol 20:253-300 (2004)). B-Ia B cells and their counterparts lacking CD5 (B-Ib) are found most abundantly in the pleural and peritoneal cavities and variably constitute 1-2% of B cells in the spleen. Bl B cells spontaneously produce quantities of natural IgM antibodies that often exhibit polyspecificity and weak autoreactivity. The combination of natural antibodies and the intrinsically facile response of MZ B cells together constitute an important first line of defense against infection, particularly by encapsulated bacteria, before FM B cells responses mature (Martin et al., Immunity, 14:617-629 (2001)). Based on early observations that cells obtained from fetal liver, but not adult bone marrow, can reconstitute the Bl B cell population after adoptive transfer into irradiated mice, Bl B cells have been considered a lineage that is distinct from conventional adult (B2) B cells. However, several more recent lines of evidence support an alternative model postulating that Bl B cell differentiation can be promoted by BCR signaling driven by cross-linking with T cell-independent antigen.

Many aspects of these processes of testing for self-reactivity and receptor editing, including the development stage at which they occur and their immunological significance, remain enigmatic and controversial. One difficulty in examining the developmental staging of these processes in a normal immunological context is that the RAG proteins are required for initial assembly of the genes encoding the B cell receptor. Hence, when either RAG protein is functionally null, B cell development is arrested well before any antigen receptors, self-reactive or otherwise, are expressed on the B cell surface. Thus, studies of receptor editing/revision have relied on genetically engineered mice carrying rearranged antigen receptor genes. Because the genes are already rearranged, lymphocyte development may be unnaturally accelerated and immune repertoires display restricted specificity and limited diversity.

SUMMARY OF THE INVENTION

The present invention includes transgenic animals, such as transgenic mice, that express a dominant-negative form of RAG-I during the transitional stage of B lymphocyte development, permitting initial antigen receptor gene rearrangement but blocking receptor editing and/or receptor revision that occurs during later periods in development. In these transgenic mice, B cells exhibiting a transitional phenotype accumulate in the periphery, but not in primary lymphoid organs of the transgenic mice. The transgenic mice are partially immunodeficient, because they have less circulating IgM and IgG antibody than normal littermates. Splenocytes from the transgenic mice are also less responsive to antigenic stimulation. The phenotypes observed in the transgenic mice are reproducible with 100% penetrance in the selected founder lines.

In one aspect the present invention provides a transgenic mouse including in its genome an exogenous polynucleotide. The exogenous polynucleotide includes a coding sequence encoding a catalytically defective RAG-I polypeptide, or an analog thereof, having an amino acid sequence with at least 80% similarity to SEQ ID NO:2. The mouse exhibits less serum IgG at 4 weeks of age as compared to a wild-type littermate, and also contains mature B cells and T cells. For instance, the amount of serum IgG exhibited by the mouse at 4 weeks of age may be reduced at least 3-fold as compared to a wild-type littermate. The exogenous polynucleotide may include a promoter operably linked to the coding sequence, and the promoter may be a tissue specific promoter that is expressed in lymphoid lineage cells, such as B cells. The amino acids of the RAG-I polypeptide, or analog thereof, corresponding to amino acids 600, and 708, and 962 of SEQ DD NO:2 are each independently alanine, glycine, serine, threonine, or proline. The transgenic mouse may be chimeric for the exogenous polynucleotide. The transgenic mouse may be heterozygous for the exogenous polynucleotide. Also provided by the present invention is a cell obtained from the transgenic mouse, wherein the cell includes the exogenous polynucleotide.

In another aspect the present invention provides a transgenic mouse including in its genome an exogenous polynucleotide, wherein the exogenous polynucleotide includes a nucleotide sequence having at least 80% similarity to SEQ ID NO:1 and encodes a catalytically defective RAG-I polypeptide, or an analog thereof. The mouse exhibits less serum IgG at 4 weeks of age as compared to a wild-type littermate, and also contains mature B cells and T cells. For instance, the amount of serum IgG exhibited by the mouse at 4 weeks of age may be reduced at least 3-fold as compared to a wild-type littermate. The exogenous polynucleotide may include a promoter operably linked to the coding sequence, and the promoter may be a tissue specific promoter that is expressed in lymphoid lineage cells, such as B cells. The transgenic mouse may be chimeric for the exogenous polynucleotide. The transgenic mouse may be heterozygous for the exogenous polynucleotide. Also provided by the present invention is a cell obtained from the transgenic mouse, wherein the cell includes the exogenous polynucleotide.

In yet another aspect, the present invention provides a method for making a transgenic mouse. The method includes introducing into a fertilized mouse egg an exogenous polynucleotide including a coding sequence encoding a catalytically defective RAG-I polypeptide, or an analog thereof, with an amino acid sequence having at least 80% similarity to SEQ ID NO:2, and implanting in a female mouse the fertilized mouse egg including the exogenous polynucleotide to produce a chimeric mouse, wherein the chimeric mouse includes in a germ cell the exogenous polynucleotide.

In another aspect the present invention provides a transgenic mouse including cells expressing a catalytically defective RAG-I polypeptide, or an analog thereof, with an amino acid sequence having at least 80% similarity to SEQ ID NO: 1. The mouse exhibits less serum IgG at 4 weeks of age as compared to a wild-type littermate, and also contains mature B cells and T cells. For instance, the amount of serum IgG exhibited by the mouse at 4 weeks of age may be reduced at least 3-fold as compared to a wild-type littermate. The exogenous polynucleotide may include a promoter operably linked to the coding sequence, and the promoter may be a tissue specific promoter that is expressed in lymphoid lineage cells, such as B cells. The amino acids of the RAG-I polypeptide, or analog thereof, corresponding to amino acids 600, and 708, and 962 of SEQ ID NO:2 are each independently alanine, glycine, serine, threonine, or proline. The transgenic mouse may be chimeric for the exogenous polynucleotide. The transgenic mouse may be heterozygous for the exogenous polynucleotide. Also provided by the present invention is a cell obtained from the transgenic mouse, wherein the cell includes the exogenous polynucleotide.

BRIEF DESCRIPTION OF THE FIGURES

Figure IA illustrates a transgene (Tg). Figure IB depicts a nucleotide sequence (SEQ ID NO:1) encoding a catalytically defective RAG-I polypeptide and the amino acid sequence (SEQ ID NO:2) encoded by the nucleotides. The underlined nucleotides in Figure IB encode the amino acids of the DDE triad, and the locations of the amino acids of the DDE triad are underlined.

Figure 2 is a Southern blot analysis of genomic DNA from Tg founders. Tg copy number and founder ID are shown above the blot.

Figure 3 illustrates levels of RAG-I (Rl) transcript detected in various organs from Tg mice derived from founders #1 (TgFl) and #15 (TgFl 5). Levels were determined using real-time PCR, and are shown relative to normal mice.

Figure 4 illustrates specific detection Rl Tg or beta-actin transcript in spleen. Tg-specific PCR profiles obtained from RNA before reverse transcription (RT) are similar to those obtained after RT from samples prepared from normal mice. Figure 5 illustrates an antibody panel for analysis of lymphocyte populations.

Figures 6A-6E are FACS plots obtained from Tg and normal (Tg-) mice. Figure 6A, single cell suspensions were prepared from spleen, bone marrow (top), thymus (bottom), and mesenteric lymph node from a 5 month-old transgenic mouse (Tg+) and its normal littermate (Tg-) and stained with antibodies to CD19 and B220 (left) or CD4 and CD8 (right). Figure 6B, splenocytes from a 4 week-old transgenic mouse and its normal littermate were stained with antibodies to CD 19 (FITC or PE) and B220 (APC or Biotin-PerCP). B22θ'° and B220^hl CD 19⁺ populations were gated and analyzed for forward and side scatter profiles, and expression of slgM, slgD, CD21, CD23, CD24, CD44, and CD93. Profiles are representative of 3 Tg+ animals and 2 Tg- animals examined (all littermates). Figure 6C, peritoneal cells and splenocytes were stained with antibodies to CD 19 and B220, and B220¹⁰ and B220^hi CD19⁺ populations were gated and analyzed for expression of IgM and CD5. Figure 6D, bone marrow preparations from 4 week-old mice were stained with CD 19 and B220; CD19⁺B220⁺ cells were analyzed for expression of slgD and slgM. Figure 6E, cell suspensions from spleen (top) or thymus (bottom) were stained with antibodies to CD 19 and B220 (top) or CD4 and CD8 (bottom). H-2Kb expression (histograms) in gated cell populations from Tg+ and Tg- mice were compared (right).

Figure 7 illustrates serum IgG and IgM concentrations (n=7 pairs).

Figure 8 illustrates the response of splenocytes to LPS stimulation or anti- IgM cross-linking using MTT assay (n=3).

Figures 9A-9E illustrate PCR-based immunoglobulin repertoire analysis, revealing similar gene segment usage in transgenic and normal mice, except for VHQ52. Four-fold serially diluted genomic DNA from splenocytes of non- transgenic littermate mice were amplified for VH and DJK rearrangement with a degenerative 5' VH { VHJ558 (FIG.9A), VH7183 (FIG. 9B), and VHQ52 (FIG. 9C) } and 3' JH4 primer sets. DH to JH was amplified using degenerative 5' DH and 3' JH4 primers (FIG. 9D). Vk to Jk was amplified with a 5' degenerative Vk and a 3' Jk5 primer (FIG. 9E). PCR products were detected by Southern blot hybridization using 32P labeled oligo-probe specific for JH4 and Jk5 regions. DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS OF THE

INVENTION

The present invention includes a transgenic animal, for instance, a transgenic mouse, having in its genome an exogenous polynucleotide. As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides, and includes both double- and single-stranded DNA and RNA. A polynucleotide may include nucleotide sequences having different functions, including for instance coding sequences, and non-coding sequences such as regulatory sequences. A polynucleotide can be obtained directly from a natural source, or can be prepared with the aid of recombinant, enzymatic, or chemical techniques. A polynucleotide can be linear or circular in topology. A polynucleotide can be, for example, a portion of a vector, such as an expression or cloning vector, or a fragment. An "exogenous polynucleotide" refers to a foreign polynucleotide, i.e., a polynucleotide that is not normally present in a cell of an animal, or a polynucleotide that is normally present in a cell of an animal, but is operably linked to a regulatory region to which it is not normally operably linked. A regulatory sequence is a nucleotide sequence that regulates expression of a coding region to which it is operably linked. Nonlimiting examples of regulatory sequences include promoters, enhancers, transcription initiation sites, translation start sites, translation stop sites, and terminators. "Operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A regulatory sequence is "operably linked" to a coding region when it is joined in such a way that expression of the coding region is achieved under conditions compatible with the regulatory sequence. A "coding region" is a nucleotide sequence that encodes a polypeptide and, when placed under the control of appropriate regulatory sequences, expresses the encoded polypeptide. The boundaries of a coding region are generally determined by a translation start codon at its 5' end and a translation stop codon at its 3' end.

The exogenous polynucleotide present in a transgenic animal of the present invention includes a coding region encoding a catalytically defective RAG-I polypeptide, or an analog thereof. Typically, the exogenous polynucleotide also includes one or more regulatory regions operably linked to the coding region. As used herein, the term "polypeptide" refers broadly to a polymer of two or more amino acids joined together by peptide bonds. The term "polypeptide" also includes molecules which contain more than one polypeptide joined by a disulfide bond, or complexes of polypeptides that are joined together, covalently or noncovalently, as multimers (e.g., dimers, tetramers). Thus, the terms peptide, oligopeptide, and protein are all included within the definition of polypeptide and these terms are used interchangeably. It should be understood that these terms do not connote a specific length of a polymer of amino acids, nor are they intended to imply or distinguish whether the polypeptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring. The present invention also includes the exogenous polynucleotide, and the polypeptide it encodes.

As used herein, the term "catalytically defective RAG-I polypeptide" is a polypeptide that is unable to catalyze the nicking and transesterification steps of V(D)J recombination, has DNA binding activity, and has structural similarity with a wild-type RAG-I polypeptide. Further, expression of a "catalytically defective RAG-I polypeptide" in a transgenic mouse as described herein will result in a mouse described in the Examples, i.e., a mouse having a B cell population that is CD45R(B220)^loCD19⁺, and further having other phenotypes such as a reduction in overall serum immunoglobulin levels, reduced levels of serum IgM and IgG, partial immunodeficiency, and decreased responsiveness to antigenic stimulation. Each of these characteristics is described herein. An exemplary catalytically defective RAG-I polypeptide useful in the present invention includes the amino acid sequence depicted at SEQ ID NO:2 (Fig. IB).

Wild-type RAG-I polypeptides are known in the art (see, for instance, the amino acid sequence disclosed at Genbank accession number NM_009019 and NP_033045.1), and catalyze the nicking and transesterification steps of V(D)J recombination (McBlane et ah, Cell, 83:387-395 (1995)). In contrast to wild- type RAG-I polypeptides, a catalytically defective RAG-I polypeptide encoded by the exogenous polynucleotide of the present invention is catalytically defective, i.e., it is unable to catalyze the nicking and transesterification steps of V(D)J recombination at the same levels as a wild-type RAG-I polypeptide. Methods for determining whether a RAG-I polypeptide is catalytically defective are described herein. Methods for determining whether a RAG-I polypeptide has a DNA binding activity are also described herein.

A wild-type RAG-I polypeptide can be rendered catalytically defective by the presence of various mutations. For instance, a set of three carboxylate amino acids (amino acids having side chains containing a carboxyl group) has been identified as playing a role in the catalytic activity of RAG-I polypeptides (Fugmann et al., MoI. Cell 5:97-107 (2000), Kim et al., Genes Dev. 13:3070- 3080 (1999), and Landree et al., Genes Dev. 13:3059-3069 (1999)). These amino acids, often referred to as a DDE triad, are the aspartate at residue 600, the aspartate at residue 708, and the glutamate at residue 962 of a murine RAG-I polypeptide. Accordingly, a catalytically defective RAG-I polypeptide can include a mutation of one, two, or all three of the amino acids corresponding to a DDE triad. For instance, a catalytically defective RAG-I polypeptide can include a mutation of the aspartate corresponding to residue 600 of the wild-type RAG-I polypeptide, the aspartate corresponding to residue 708 of the wild-type RAG-I polypeptide, the glutamate corresponding to residue 962 of the wild-type RAG-I polypeptide, or a combination thereof. In some aspects, a catalytically defective RAG-I polypeptide includes a mutation at each of the three residues. A mutation may be the non-conservative substitution of an aspartate or a glutamate for an amino acid that does not contain a side chain with a carboxyl group, such as Asn, GIn, Ala, GIy, Pro, Cys, Ser, Thr, Tyr, Arg, Lys, Ue, VaI, Leu, Met, Phe, Trp, or His. In one aspect an exogenous polynucleotide encodes a catalytically defective RAG-I polypeptide having an alanine at the aspartate present at residue 600 of the wild-type RAG-I polypeptide, the aspartate at residue 708 of the wild-type RAG-I polypeptide, and the glutamate at residue 962. An example of such a RAG-I polypeptide is depicted at SEQ ID NO:2.

A catalytically defective "analog" of a RAG-I polypeptide includes a catalytically defective RAG-I polypeptide that has been modified by the addition, substitution, or deletion of one or more contiguous or noncontiguous amino acids, as long as the analog retains DNA binding activity and the ability to produce a transgenic mouse with a B cell population that is CD45R(B220)'°CD19⁺, and further having other phenotypes as described herein. An analog can thus include additional amino acids at one or both of the termini of a RAG-I polypeptide, deletions of amino acids at one or both of the termini of a RAG-I polypeptide, or a combination thereof. For example, a catalytically defective RAG-I polypeptide may include a deletion of one or more consecutive amino acids from the amino terminal end, and up to a deletion of the first 388 amino acids. Likewise, a catalytically deflective RAG-I polypeptide may include a deletion of one or more consecutive amino acids from the carboxy terminal end, and up to a deletion of the last 31 amino acids.

Substitutes for an amino acid in the RAG-I polypeptides useful herein are preferably conservative substitutions, which are selected from other members of the class to which the amino acid belongs. For example, it is well-known in the art of protein biochemistry that an amino acid belonging to a grouping of amino acids having a particular size or characteristic (such as charge, hydrophobicity and hydrophilicity) can generally be substituted for another amino acid without substantially altering the structure of a polypeptide. For the purposes of this invention, conservative amino acid substitutions are defined to result from exchange of amino acids residues from within one of the following classes of residues: Class I: Ala, GIy, Ser, Thr, and Pro (representing small aliphatic side chains and hydroxyl group side chains); Class II: Cys, Ser, Thr and Tyr (representing side chains including an -OH or -SH group); Class III: GIu, Asp, Asn and GIn (carboxyl group containing side chains): Class IV: His, Arg and Lys (representing basic side chains); Class V: He, VaI, Leu, Phe and Met (representing hydrophobic side chains); and Class VI: Phe, Trp, Tyr and His (representing aromatic side chains).

Preferred catalytically defective analogs of RAG-I include those analogs that are at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96%, at least 97%, at least 98%, or at least 99% identical to the RAG-I polypeptide depicted at SEQ ID NO:2. Such analogs contain one or more amino acid deletions, insertions, and/or substitutions relative to the RAG-I polypeptide depicted at SEQ ID NO:2. Several conserved domains are known to be present in a wild-type RAG-I polypeptide, including the basic domain, the ring finger domain, the zinc finger domain, the nonamer binding domain, and the coding flank binding domain. Typically, these domains are conserved in a catalytically defective RAG-I polypeptide and analogs thereof.

Percent identity between two polypeptide sequences is generally determined by aligning the residues of the two amino acid sequences to optimize the number of identical amino acids along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of identical amino acids, although the amino acids in each sequence must nonetheless remain in their proper order. Preferably, two amino acid sequences are compared using the BESTFIT algorithm in the GCG package (version 10.2, Madison WI), or the Blastp program of the BLAST 2 search algorithm, as described by Tatusova, et al. (FEMS Microbiol Lett 1999, 174:247- 250), and available through the World Wide Web, for instance at the internet site maintained by the National Center for Biotechnology Information, National Institutes of Health. Preferably, the default values for all BLAST 2 search parameters are used, including matrix = BLOSUM62; open gap penalty = 11, extension gap penalty = 1, gap x_dropoff = 50, expect = 10, wordsize = 3, and optionally, filter on. In the comparison of two amino acid sequences using the BLAST search algorithm, structural similarity is referred to as "identity."

Methods for determining whether a RAG-I polypeptide is catalytically defective are known to the art and routine (see, for instance, Fugmann et al., MoI. Cell 5:97-107 (2000), Kim et al., Genes Dev. 13:3070-3080 (1999), and Landree et al., Genes Dev. 13:3059-3069 (1999)). The catalytic activity of a RAG-I polypeptide can be tested for the ability to support V(D)J recombination using a cell-culture based assay in which expression vectors encoding RAG-I and RAG- 2 are cotransfected with a reporter plasmid V(D)J recombination substrate. In vitro assays can also be used (McBlane et al., Cell 83:387-395 (1995)), and typically include incubation of purified RAG-I, RAG-2, and a radiolabeled model recombination signal sequence, or RSS) substrate in the presence of an appropriate buffer containing either MgCl₂ or MnCl₂. After incubation the cleavage products are resolved by gel electrophoresis and visualized by autoradiography. A decrease in the ability of a mutant RAG-I polypeptide to cleave the RSS substrate when incubated with a wild-type RAG-2 polypeptide indicates the RAG-I polypeptide is catalytically defective, provided that the RAG-I and RAG-2 proteins retain DNA binding activity, as described herein. A mutant RAG-I polypeptide is considered to be catalytically defective if the ability to cleave a substrate is reduced by at least 40-fold, at least 50-fold, or at least 60-fold relative to the wild-type RAG-I polypeptide disclosed at Genbank accession number NP_033045.1. In some aspects, a RAG-I polypeptide has no detectable catalytic activity in the absence of a RAG-2 polypeptide.

The RAG-I polypeptide encoded by the exogenous polynucleotide may optionally include a DNA binding activity. Methods for determining whether a RAG-I polypeptide has DNA binding activity are known to the art and routine. Binding reactions are typically conducted in vitro, and may be assembled similar to the cleavage assay described above, except that MgCl₂ (or MnCl₂) is replaced with CaCl₂. After incubation of the components to allow formation of a protein- DNA complex, the reaction is subjected to electrophoresis. A RAG-I polypeptide is considered to be have DNA binding activity if the mobility of the RSS substrate is decreased relative to the RSS substrate not incubated with a RAG-I polypeptide. Addition of a RAG-2 polypeptide can further reduce the mobility of the protein-DNA complex containing a RAG-I polypeptide.

A catalytically defective RAG-I polypeptide and analogs thereof will result in a transgenic mouse having a B cell population that is CD45R(B220)^loCD19⁺, and further having other phenotypes such as a reduction in overall serum immunoglobulin levels, reduced levels of serum IgM and IgG, partial immunodeficiency, and decreased responsiveness to antigenic stimulation. Whether a candidate catalytically defective RAG-I polypeptide will result in such a transgenic mouse can be tested by replacing the nucleotides of SEQ ID NO: 1 present in the construct described in Example 1 with nucleotides encoding the candidate catalytically defective RAG-I polypeptide. A candidate catalytically defective RAG-I polypeptide is the catalytically defective RAG-I polypeptide being evaluated. The resulting exogenous polynucleotide can be used to make a transgenic mouse as described herein, and then tested as described herein to determine if the transgenic mouse has a B cell population that is CD45R(B220)^loCD19⁺, and further has other phenotypes as described herein.

Polynucleotides encoding a catalytically defective RAG-I polypeptide include a polynucleotide encoding the amino acid sequence depicted at SEQ ID NO:2. An example of such a polynucleotide is shown at SEQ ID NO: 1. Also included are polynucleotides having a nucleotide sequence that is "substantially complementary" to a nucleotide sequence that encodes the amino acid sequence depicted at SEQ ID NO:2. "Substantially complementary" polynucleotides can include at least one base pair mismatch, however the two polynucleotides will still have the capacity to hybridize. For instance, the middle nucleotide of each of the two DNA molecules 5 -AGCAAATAT and 5 -ATATATGCT will not base pair, but these two polynucleotides are nonetheless substantially complementary as defined herein. Two polynucleotides are substantially complementary if they hybridize under hybridization conditions exemplified by 2X SSC (SSC: 15OmM NaCl, 15 mM trisodium citrate, pH 7.6) at 55⁰C. Substantially complementary polynucleotides for purposes of the present invention preferably share at least one region of at least 20 nucleotides in length which shared region has at least 60% nucleotide identity, preferably at least 80% nucleotide identity, more preferably at least 90% nucleotide identity and most preferably at least 95% nucleotide identity. Particularly preferred substantially complementary polynucleotides share a plurality of such regions.

"Substantially complementary polynucleotides" also includes the class of polynucleotides that encode the polypeptide having the amino acid sequence depicted at SEQ ID NO:2 as a result of the degeneracy of the genetic code. For example, the nucleotide sequence depicted at SEQ ID NO:1 is but one member of the class of nucleotide sequences that encodes a polypeptide having amino acid SEQ ID NO:2. The class of nucleotide sequences that encode a selected polypeptide sequence is large but finite, and the nucleotide sequence of each member of the class can be readily determined by one skilled in the art by reference to the standard genetic code, wherein different nucleotide triplets (codons) are known to encode the same amino acid.

Percent identity between two polynucleotide sequences is generally determined by aligning the residues of the two nucleotide sequences to optimize the number of identical nucleotides along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of identical nucleotides, although the nucleotides in each sequence must nonetheless remain in their proper order. Preferably, two nucleotide sequences are compared using the BESTFIT algorithm in the GCG package (version 10.2, Madison WI), or the the Blastn program of the BLAST 2 search algorithm, as described by Tatusova, et al. (FEMS Microbiol Lett 1999, 174:247-250), and available through the World Wide Web, for instance at the internet site maintained by the National Center for Biotechnology Information, National Institutes of Health. Preferably, the default values for all BLAST 2 search parameters are used, including reward for match = 1, penalty for mismatch = -2, open gap penalty = 5, extension gap penalty = 2, gap x_dropoff = 50, expect = 10, wordsize = 11, and optionally, filter on. Locations and levels of nucleotide sequence identity between two nucleotide sequences can also be readily determined using CLUSTALW multiple sequence alignment software (J. Thompson et al., Nucl. Acids Res., 22:4673-4680 (1994)), available at from the world wide web. Preferred polynucleotides have a nucleotide sequence encoding a catalytically defective RAG-I polypeptide or analog thereof. Such polynucleotides include those that are at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:1. The underlined nucleotides at SEQ ID NO:1 in Figure IB encode the amino acids of the DDE triad. The skilled person will recognize that these codons can be any codon that encodes the amino acid alanine.

Optionally, the exogenous polynucleotide further includes nucleotides encoding a splice donor, and such nucleotides are present at the 3' end of the coding region encoding the catalytically defective RAG-I polypeptide and operably linked to the coding region. A splice donor site is a nucleotide sequence that is generally involved in RNA splicing to remove intronic RNA sequences. Splice donor sites typically end in GT (or GU) dinucleotides. Splice donor sequences are known in the art, and can be readily obtained from genes at a position between the exon and intron where they mediate splicing. Alternately, splice donor sites may be chemically or enzymatically synthesized. Whether a polynucleotide functions as a splice donor can be easily determined using methods known in the art. An example of a splice donor site is disclosed at Genbank Accession number NM_000518 (see also Lawn et al., Cell, 21:647-651 (1980)).

The exogenous polynucleotide present in a transgenic animal of the present invention includes a promoter operably linked to the coding region encoding the catalytically defective RAG-I polypeptide or analog thereof. Promoters act as regulatory signals that bind RNA polymerase in a cell to initiate transcription of a downstream (3' direction) coding region. The invention is not limited by the use of any particular promoter, and a wide variety are known (see, for instance, Olson and Nicol, U.S. Patent 6,924,415). The promoter used in the invention can be a constitutive or an inducible promoter. In some aspects, the promoter is tissue specific. A tissue specific promoter is capable of driving transcription of a coding region in one tissue or cell while remaining largely "silent" in other tissue or cell types. It will be understood, however, that tissue specific promoters may have a detectable amount of "background" or "base" activity in those tissues where they are silent. The degree to which a promoter is selectively activated in a target tissue or cell can be expressed as an expression ratio (expression of an operably linked coding region in a target tissue/ expression of the operably linked coding region in a control tissue). In this regard, a tissue specific promoter useful in the practice of the present invention typically has an expression ratio of greater than 5, greater than 15, or greater than 25. Tissue specific promoters also include promoters that are active in one group of tissues or group of cells, and less active or silent in another group. Tissue specific promoters may be derived, for example, from promoter regions of genes that are differentially expressed in different tissues. For example, a variety of promoters have been identified which are suitable for up regulating expression in lymphoid lineage cells, such as B cells and T cells, including CD5 positive cells. Included, for example, are promoters operably linked to genes present in the MHC region of mice (e.g., the H2-K gene), the HLA region of humans, and the CD 19 gene. An exemplary promoter is the mouse MHC class I H-2K^b promoter (Genbank accession number Ml 1847, see also Kimura et al., Cell, 44:261-272 (1986)). Such promoters may be chemically or enzymatically synthesized. The skilled person will recognize that some changes to the nucleotide sequence of a promoter can be made that will have little if any effect on the promoter activity. Optionally, a promoter, including a tissue specific promoter, can include a portion of an exon. For instance, in those aspects of the invention where a tissue specific promoter is used, nucleotides normally 3' of the promoter and encoding the first amino acids of the polypeptide may be included.

The exogenous polynucleotide present in a transgenic animal of the present invention may optionally include an enhancer operably linked to the promoter. An "enhancer" is a regulatory sequence that increases the rate of transcription initiation of a coding region. Enhancers usually exert their effect regardless of the distance, upstream or downstream location, or orientation of the enhancer relative to the start site of transcription. The invention is not limited by the use of any particular enhancer, and a wide variety are known (see, for example, Blackwood et al., Science, 281:60 (1998), and Olson and Nicol, U.S. Patent 6,924,415). In some aspects, the enhancer is tissue specific. A tissue specific enhancer is capable of increasing transcription of a coding region in one tissue or cell and being largely "silent" in other tissue or cell types. It will be understood,, however, that tissue specific enhancers may have a detectable amount of "background" or "base" activity in those tissues where they are silent. The degree to which an enhancer selectively increases expression in a target tissue or cell can be expressed as an enhanced expression ratio (expression in a target tissue of a coding region operably linked to an enhancer/ expression in the target tissue of the coding region not operably linked to the enhancer). In this regard, a tissue specific enhancer useful in the practice of the present invention typically has an enhancer expression ratio of greater than 5, greater than 15, or greater than 25. Tissue specific enhancers also include enhancers that are active in one group of tissues or group of cells, and less active or silent in another group. Tissue specific enhancers may be derived, for example, from genes that are differentially expressed in different tissues. For example, a variety of enhancers have been identified which are suitable for up regulating expression in lymphoid lineage cells, such as B cells, including CD5 positive cells. Included, for example, are enhancers operably linked to genes expressed at higher levels in lymphoid lineage cells, such as enhancers associated with immunoglobulin genes. An exemplary promoter is the mouse IgH enhancer (Genbank accession number V01524, Banerji et al., Cell, 33:729-740 (1983)). Such enhancers may be chemically or enzymatically synthesized. The skilled person will recognize that some changes to the nucleotide sequence of an enhancer can be made that will have little if any effect on the enhancer activity.

The exogenous polynucleotide may be present in a vector. A vector is a replicating polynucleotide, such as a plasmid, phage, or cosmid, to which another polynucleotide may be attached so as to bring about the replication of the attached polynucleotide. Construction of vectors containing a polynucleotide of the invention employs standard ligation techniques known in the art. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989) or Ausubel, R.M., ed. Current Protocols in Molecular Biology (1994). A vector can provide for further cloning (amplification of the polynucleotide), i.e., a cloning vector, or for expression of the polypeptide encoded by the coding region, i.e., an expression vector. The term vector includes, but is not limited to, plasmid vectors, viral vectors, cosmid vectors, or artificial chromosome vectors. Typically, a vector is capable of replication in a bacterial host, for instance E. coli, a eukaryotic host such as a mouse cell, or both. Preferably the vector is a plasmid.

The present invention includes transgenic animals that contain the exogenous polynucleotide described hereinabove, and methods of making such transgenic animals. A transgenic mouse may be homozygous or heterozygous for the exogenous polynucleotide. Also included in the present invention are cells containing the exogenous polynucleotide described hereinabove. Techniques for the preparation of transgenic animals are known in the art. Exemplary techniques are described in Hammer and Taurog, U.S. Pat. No. 5,489,742 (transgenic rats); Wagner and Hoppe, U.S. Pat. No. 4,873,191, Leder and Stewart, U.S. Pat. No. 4,736,866, Mintz, U.S. Pat. No. 5,550,316, Bradley et al., U.S. Pat. No. 5,614,396, Bennett et al., U.S. Pat. No. 5,625,125, and Bernstein and Uitto, U.S. Pat. No. 5,648,061 (transgenic mice); Seamark and Wells, U.S. Pat. No. 5,573,933 (transgenic pigs); Bosselman et al., U.S. Pat. No. 5,162,215 (transgenic avian species), and Deboer et al., U.S. Pat. No. 5,741,957 (transgenic bovine species). With respect to a representative method for the preparation of a transgenic mouse, an exogenous polynucleotide of the present invention is injected into fertilized mouse eggs. The injected eggs are implanted in pseudopregnant females and are grown to term to provide transgenic mice whose cells express a catalytically defective RAG-I polypeptide encoded by the introduced exogenous polynucleotide.

A polynucleotide for random integration need not include regions of homology to mediate recombination. Where homologous recombination is desired, the introduced polynucleotide will typically include regions of homology to the target nucleotides present in the animal. Conveniently, markers for positive and negative selection may be included. Such positive and negative markers are known in the art and readily available (see, for instance, Capecchi and Thomas, U.S. Patent No. 5,464,764). For various techniques for transfecting mammalian cells, see Keown et al., (Methods Enzymol., 185:527-537 (1990). For embryonic stem (ES) cells, an ES cell line can be employed, or embryonic cells can be obtained freshly from a host, e.g. mouse, rat, guinea pig, etc. Such cells may be grown on an appropriate fibroblast-feeder layer or grown in the presence of appropriate growth factors. When ES cells have been transformed, they can be used to produce transgenic animals. After transformation, the cells may be plated onto a feeder layer in an appropriate medium. Cells containing the introduced polynucleotide can be detected by employing a selective medium. After sufficient time to allow colonies to grow, they are picked and analyzed for the occurrence of homologous recombination or integration of the introduced polynucleotide. Those colonies that are positive can then be used for embryo manipulation and blastocyst injection. Blastocysts can be obtained from 4 to 6 week old normally mated or superovulated females. The ES cells are usually trypsinized, and the modified cells are injected into the blastocoel of the blastocyst. After injection, the blastocysts are returned to each uterine horn of pseudopregnant females. Females are then allowed to proceed to term and the resulting litters are screened for the presence of the introduced polynucleotide. The resulting animals, typically chimeric animals, are screened for the presence of the introduced polynucleotide and may be used to produce heterozygous or homozygous progeny.

Heterozygous progeny, such as heterozygous mice, have the characteristics of accumulating a B cell population that is abundant in the spleen. This B cell population is detectable in peripheral blood, bone marrow, and lymph nodes as the animal ages. For instance, when the animal is a mouse, this B cell population is typically detectable in peripheral blood, bone marrow, and lymph nodes at the age of 4 weeks. The B cell population is CD45R(B220)'°CD19⁺. These cells, when obtained from spleens at 4 weeks of age, are also IgM^hiIgD^l0CD21 ^loCD23 ^/loCD24^intCD44^hiCD62U^/IoAA4. rCD5^l0H2Kb^hi. Methods for identifying and measuring expression of these cell surface markers can be easily accomplished by the skilled person using readily available antibodies and methods. Typically, the antibodies are conjugated to a detectable marker, such as a fluorochrome. The presence and amount of an antibody associated with a cell can be determined using flow cytometry methodologies. The descriptors "lo," "int," and "hi" refer to low, intermediate, and high levels of expression as determined by brightness. Brightness of surface marker expression is based on where in the log scale of fluorescence the cells reside upon flow cytometric analysis in comparison with mature B cells obtained from the spleen. For instance, in normal B mature cells in the spleen the mean log fluorescence intensity of B220 expression is usually between 10²-10³ absorbance units. For comparison, the level of B220 expression in the "B220¹⁰" B cells in transgenic mice is between lO'-lO² absorbance units.

Heterozygous progeny also typically display a delayed progression of B cells in the bone marrow at the age of 4 weeks. The homozygous progeny also have a reduction in overall serum immunoglobulin levels compared to non- transgenic animals, and also have levels of serum IgM and IgG are reduced by at least 3-fold, at least 4-5-fold, or at least 6-fold relative to the levels of serum IgM and IgG observed in normal littermates. This decrease in circulating IgM and IgG results in partial immunodeficiency. The heterozygous progeny are also display a non-responsiveness to antigenic stimulation. Whether an animal is non- responsive can be determined by obtaining a population of B cells, for instance, from the spleen, exposing the cells to an antigen, and measuring activation of the cells. Cells from a transgenic animal that is homozygous for an exogenous polynucleotide will be at least 3-fold, at least 4-5-fold, or at least 6 fold less responsive to an antigen that a normal littermate.

Transgenic animals expressing a catalytically defective RAG-I polypeptide, recombinant cell lines derived from such animals, and transgenic embryos can be used as a model of partial immunodeficiency due to the abnormally low production of antibody. Furthermore, these transgenic animals are expected to be useful in the study of autoimmune disease, because if presumably autoreactive B cells are prevented from undergoing receptor editing and somehow become activated, the resulting B cells may produce autoantibodies, leading to progressive autoimmune disease. A third potential application is toward the study of leukemia and lymphoma, because the accumulating B cells may be prone to malignant transformation under certain circumstances (e.g. radiation or infection), providing a potential model for peripheral B cell malignancies. For instance, it is possible that a transgenic animal expressing a catalytically defective RAG-I polypeptide will develop B- chronic lymphocytic leukemia (B-CLL) as a result of prolonged survival. CD5⁺ Bl B cells exhibit such traits as self-reactivity and self-renewal (Hardy and Hayakawa, Adv. Immunol,. 55:297-339 (1994)), features that have implicated them in the etiology of certain autoimmune diseases and a fairly common form of leukemia known as B-CLL. Thus, it is possible that the transgenic mice described here, which accumulate a B220^loCD19⁺ B cell population that also expresses low levels of CD5, may be predisposed to developing autoimmunity or cancer. Understanding what might dictate the outcome could provide insight into the etiology of human systemic lupus erythematosus (SLE) and B-CLL, where B 1 B cells have been implicated in disease pathogenesis. Both SLE and B-CLL are clinically significant diseases in the United States. According to the Lupus Foundation of America, approximately 1,500,000 Americans have a form of SLE; 90% of individuals diagnosed with the disease are women, and 80% of those afflicted with systemic lupus develop it between the ages of 15 and 45. According to the European Society for Medical Oncology, B-CLL has an incidence of 3/100,000 per year in the western hemisphere; after the age of 70, the incidence increases to almost 50/100,000 per year. B-CLL represents the most frequent non-Hodgkin's lymphoma (11%) and leukemia of adults (25%). Since B-CLL is thought to often arise from CD5+ Bl B cell precursors, it is perhaps not surprising that autoimmunity is prevalent among patients with B-CLL, particularly autoimmune cytopenias. Paradoxically, B-CLL patients often develop hypogammaglobulinemia as the disease progresses, perhaps because the accumulating B cell population interferes directly or indirectly with cognate interactions between normal B and T cells (see Caligaris-Cappio and Hamblin, J. Clin. Oncol,. 17:399-408 (1999)). In this respect, the transgenic animals described herein may serve as a useful model for progressive hypogammaglobulinemia associated with an accumulating Bl -like B cell population.

The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein. Example 1 Generation of RAG-I (DDE->A) transgenic mice

Transgenic mice encoding a form of full-length RAG-I in which all three active site residues are substituted by alanine (DDE->A) were produced to determine if RAG-I DDE mutants might suppress endogenous V(D)J recombination activity in a dominant negative fashion when the two forms of RAG-I are coexpressed in vivo.

Production of the DNA construct. All RAG-I mutants were generated in pBluescript containing the core RAG-J cDNA sequence by site-directed mutagenesis using recombination PCR as described (Jones and Winistorfer, Biotechniques 12:528-530, 532, 534-525 (1992)) and verified by DNA sequencing. The following primer pairs were used for PCR to generate the RAG- 1 mutants: D600AFor: GTAAAGGAGTCTTGCGCAGGAATGGGGGATGTG (SEQ ID NO:9), DόOOARev:

CACATCCCCCATTCCTGCGCAAGACTCCTTTAC (SEQ ID NO: 10), D708AFor: CTTCAGGGGCACCGGTTACGCTGAAAAACTTGTCC (SEQ ID NO: 11), D708ARev: GGACAAGTTTTTCAGCGTAACCGGTGCCCCTGAAG (SEQ ID NO: 12), E962AFor:

GCAAGTGAGGGAAATGCATCGGGTAACAAGCTG (SEQ ID NO: 13), and E962ARev: CAGCTTGTTACCCGATGCATTTCCCTCACTTGC (SEQ ID NO: 14).

Single mutants were generated initially. Next, a D600A/D708A mutant was generated by sequential rounds of site-directed mutagenesis using pBluescript encoding RAG-I D600A as a PCR template. The D600A/D708A RAG-I sequence was subcloned into the mammalian expression vector pcDNAl containing a full-length RAG-J cDNA (pcRAGl) described by Lin and Desiderio (Lin and Desiderio, Science, 260:953-959 (1993)) by cassette replacement using BsrGl, generating pcRAGl D600A/D708A. The vector pcRAGl D600A/D708A/E962A was generated by subcloning a BspLUl U-BstZlll fragment containing the E962A RAG-J sequence into pcRAGl D600A/D708A.

The mDDE RAG-I transgene construct encoding the RAG-1(DDE->A) mutant (also referred to as the mDDE RAG-I transgene) was generated by inserting a BamHΪ fragment from pcRAGl D600A/D708A/E962A containing the mutant RAG-I cDNA into the BamEl site of pHSE3' described by Pircher et al ( Pircher et al., Embo J., 8:719-727 (1989)), generating pHSE3'mDDE RAG-I. An Xhoϊ fragment from pHSE3' mDDE RAG-I, containing the H-2K^b promoter, the full-length D600A/D708A/E962A RAG-I cDNA, the human beta-globin splice donor, and the murine immunoglobulin heavy chain enhancer, was separated from the vector by preparative agarose gel electrophoresis, recovered using the QiaQuick Gel Extraction Kit (Qiagen Sciences, MD), and eluted into 10 mM Tris, pH 8.5. Transgenic mice were generated using a commercial vendor (Xenogen Biosciences, Cranbury, NJ).

The mDDE RAG-I transgene has the following characteristics. On the 5' end of the transgene, the H-2K^b promoter sequence is found at GenBank accession #M11847. Starting with nucleotide 2011 of this sequence, the junction with the mutant RAG-I cDNA reads as follows: H-2K^b promoter: 5'...GCAGAACTCAGAAGTCG-S' (SEQ ID NO: 15), then Linker sequence: 5'- TGGTCG ACTCT AG AGGATCC AC-3' (SEQ ID NO: 16, this sequence contains Sail, Xbal, and BamHI restriction sites), then full-length mDDE RAG-I cDNA, starting at first codon 5 -ATGGCTGCC...-3'. On the 3' end of the transgene, the junction with the mutant RAG-I cDNA reads as follows: 3 -end of RAG-I: 5 '...G AGTTTTA A-3', then Linker sequence: 5 -TAGGATCTCC-S', then human beta-globin sequence, beginning at BamHI site in exon II of the gene, which can be found at Genbank accession number V00499 starting at nucleotide 580: 5 - GGATCCTGAG...-3 and contains the entire 3' end of the gene.

The transgenic mice were produced under our direction by Xenogen Biosciences (Cranbury, NJ). Briefly, mouse embryos were injected with the linearized DNA construct using methods disclosed in Wagner and Hoppe (U.S. Patent No. 4,873,191). The linearized DNA construct encoded a full-length RAG-1(DDE->A) mutant under the control of the H-2K^b promoter (FIG. 1) (see Pircher et al., EMBO J 8:719-727, 1989), which has been shown to support transcription through lymphocyte development in the B lineage, T lineage, or both in a founder-specific fashion (Pircher et al., EMBO J 8:719-727, 1989; Malek et al., Proc Natl Acad Sci USA 95:7351-7356, 1998). The nucleotide sequence of the DNA construct is depicted at SEQ JJD NO: 1. Selection of transgenic mice containing the transgene incorporated into the genome of the recipient mice was done using routine methods (see, for instance, Capecchi and Thomas, U.S. Patent No. 5,464,764). The transgene (Tg) was detected in genomic DNA isolated from tail snips by PCR using construct-specific primers and verified by Southern hybridization using a RAG-I BsrGΪ restriction fragment encoding residues 484-727 to detect ZtømHi-digested genomic DNA (see FIG. 2). The construct-specific primers were Η2kb-for (specific for H-2Kb promoter): GATCAGAACTCGGAGACGAC (SEQ ID NO:3), and Rl-1187REV (specific for RAG-I): ACCAGGCTTCTCTGGAACTAC (SEQ ID NO:4). The probe was a 728 base pair BsfGl fragment from the RAG-I cDNA that included nucleotides 1450 and 2178 from the RAG-I cDNA. Of the 61 animals born, 22 (36%) screened positive for the presence of the Tg by both techniques (FIG. T). The ratio between germline RAG-I and Tg-encoded RAG-I on Southern blots suggest founders carry anywhere from one to fifty copies of the Tg.

Example 2 Tg expression in RAG-1(DDE->A) Tg mice.

Understanding any phenotype observed in these Tg mice includes identifying the cell lineage(s) and developmental stage(s) in which the Tg is expressed. Tg expression may be assessed both at the transcriptional level and at the protein level, as they are not necessarily directly correlated. As the RAG-I mutant encoded by the Tg is identical to endogenous RAG-I except for the presence of three point mutations (DDE->A), the two forms of RAG-I cannot be easily distinguished. However, Tg expression should be evident by an overabundance of RAG-I protein or RNA transcript in cells or tissues of Tg mice relative to normal littermates. As disclosed herein, Tg expression has been analyzed at the transcriptional level by real-time PCR. In normal, unimmunized mice, RAG-I is expressed in select pro- and pre-B cell and CD4-CD8- and CD4+CD8+ T cell compartments found in primary lymphoid organs (e.g. bone marrow and thymus), but not in mature lymphocytes found in secondary lymphoid organs (e.g. spleen and lymph node). To examiner RAG-I transcript levels in Tg mice, total RNA was isolated from primary and secondary lymphoid organs and liver from Tg mice and normal littermates using a commercially available kit (RNAgents, Promega) that incorporates treatment with DNAse I to remove contaminating genomic DNA, and reversed transcribed into cDNA using random hexamer primers. Next, real-time PCR assays were performed using the SYBR Green I dye to detect cDNAs of RAG-I and the housekeeping gene beta- actin. PCR amplification was performed under conditions similar to those described in a recent review (Giulietti et al., Methods 25:386-401, 2001). Primer sequences for beta-actin are described by Giulietti et al. (Methods 25:386-401, 2001). RAG-I Forward primer was ATGGCTGCCTCCTTGCCGTCTACC (SEQ ID NO:5) (Hikida et al., Science 274:2092-2094 (1996)), and RAG-I Reverse primer was CTGAGGAATCCTTCTCCTTCTGTG (SEQ ID NO: 6). Single amplicons of the expected sizes were observed. RAG-I expression in Tg mice was compared to normal mice after normalizing to β-actin expression using the comparative threshold cycle (C₁) method (Giulietti et al., Methods 25:386-401 (2001)). The data show that in Tg mice derived from founder animals #1 and #15, RAG-I is more highly expressed in liver, spleen and lymph node, but, interestingly, not in bone marrow or thymus (FIG 3). A real-time PCR assay designed to specifically detect Tg transcript using primers targeting the 3' end of RAG-I and the beta-globin splice donor. The sense (forward) primer was TGGGCATTGAGGACTCTCTGGAAA (SEQ ID NO:7) and the anti-sense (reverse) primer specific for beta-globin sequence was GTCCCATAGACTCACCCTGAAGTT (SEQ ID NO:8). The real-time PCR assay results supports this conclusion, and further suggests that full-length Tg transcript is being produced (FIG 4). RAG-I expression was detected in 2 x 10⁶ thymocytes derived from both Tg and normal mice using an affinity-purified rabbit polyclonal antibody specific for RAG-I (Ab307, Lin and Desiderio, Science, 260:953-959 (1993)). However, RAG-I protein was not visualized in splenocytes obtained from Tg mice, possibly because the cells expressing RAG-I represent a small percentage of total splenocytes.

Example 3 Immunophenotyping of RAG-I (DDE->A) Tg mice

Flow cytometry is the method of choice for analyzing lymphocyte development. As such, a group of antibodies to a variety of cell type- and stage- specific surface antigens was assembled that were used to discriminate between various B and T cell populations (developed from Martensson and Ceredig, Immunology 101:435-441, 2000; Hardy et al., J Exp Med 173:1213-1225, 1991; Kincade et al., Curr Top Microbiol Immunol 251:67-72, 2000; Matsuzaki et al., J Exp Med 178:1283-1292, 1993; Tudor et al., Immunity 12:335-345, 2000; Wilson et al., J Exp Med 179:1355-1360, 1994; Godfrey and Zlotnik, Immunol Today 14:547-553, 1993; and Kincade et al., Immunol Rev 175:128-137, 2000). To discriminate the pro-from pre-B cells, the Kincaid approach was chosen over the Hardy scheme based on the brightness of CD72 relative to BP-I and HAS[CD24]). Immature, transitional (Tl -T3) and mature B cell populations can be distinguished based on expression of slgM, slgD, CD21, CD23, CD24, and CD62L (Su and Reawlings, J Immunol 168:3202-2110, 2002; Allman et al., J Immunol 167:6834-6840, 2001; Loder et al., J Exp Med 190:75-89, 1999).

Offspring from RAG-1(DDE->A) Tg founder animals #1, #15, and #45 were sacrificed at four-six weeks of age. Single-cell suspensions, depleted of red blood cells, were prepared from bone marrow, spleen, lymph node and thymus and stained with FITC-, PE-, APC-, and Per-CP-conjugated antibodies in the combinations listed in figure 5 following standard protocols available through Flow Cytometry Core Facility at Creighton University, Omaha, NE. Samples were examined on a four-color FACSCalibur and analyzed using the CellQuestPro (BD Biosciences) or FlowJo (Tree Star) software; typically, >20,000 events were collected in a lymphocyte gate. Among transgenic animals tested, two founder animals, #1 and #15, reproducibly accumulated a population of CD45R(B220)^loCD19⁺ B cells in the spleen by 2 weeks of age, and in bone marrow, lymph node and peripheral blood of older mice (Fig. 6A, left panel). These results show that CD45R(B220)^!oCD19⁺ B cells are detectable in transgenic mice by the age of two weeks and accumulate to 10% by four weeks of age. Individual animals examined beyond that time point show populations of Bl B cells ranging to almost 50% of splenic B cells. T cell subpopulations are quite similar between normal and transgenic mice (Fig. 6A, right panel). Extensive flow cytometric analysis from 4 week-old mice revealed that the B220^loCD19⁺ B cells were slightly larger and more granular than B220^hiCD19⁺ B cells from normal mice, as evidenced by their higher mean forward and side scatter properties, and were characterized as being IgM^hTgD^loCD21^l0CD23^{" /l0}CD24^intCD44^hiCD62L^"/l0AA4.r (Fig. 6B). These cells expressed low levels of CD5, comparable to peritoneal B22O¹⁰ B cells (Fig. 6C), and CD43, but did not express the germinal center marker GL-7. The B220^loCD19⁺ B cells resembled Tl, MZ and Bl B cells with respect to surface IgM and IgD expression. However, based on the expression of CD21, CD24, and CD93(AA4.1), B220^loCD19⁺ B bear little resemblance to Tl and MZ B cells. Instead, since B220^loCD19⁺ B cells share the forward and side scatter properties of Bl B cells, and display a similar staining pattern with antibodies to CD5 and B220, it was tentatively concluded that the B220^loCD19⁺ B cells observed in the transgenic mice most closely resembled the Bl B cell. At 4 weeks of age, the splenic B220^hlCD19⁺ population the transgenic mice contained fewer mature cells, and an overabundance of immature (IgM⁺IgD^") B cells relative to its counterpart population in nontransgenic mice. Consistent with this observation, most cells in this population were also CD2rCD23^"CD24^hiCD44^intCD62L^". In the bone marrow of 4 week-old transgenic mice, B220^loCD19⁺ B cells were not abundant. Interestingly, however, transgenic mice showed slightly smaller percentages of cells in the B220⁺IgM⁺ and IgM⁺IgD⁺ compartments (Fig. 6D), raising the possibility that transit through these developmental stages were delayed by transgene expression. H-2K^b expression was analyzed at various stages of B and T cell development. In splenic B22θ'°CD19⁺ B cells, H-2K^b was expressed more highly than in bone marrow B220⁺ B cells (Fig. 6E). This observation raises the possibility, as yet unproven, that mDDE RAG-I might be more highly expressed in B220^!oCD19⁺ B cells than in B cells present in the bone marrow. As a result, the dominant-negative effect may more be more strongly enforced (less leaky) at this stage than at earlier stages of B cell development. In contrast to B cells, H- 2K^b is not expressed on CD4⁺CD8⁺ T cells in the thymus (Fig. 6E), a developmental stage where RAG-I is normally expressed. This result is consistent with data that RAG-I is not overexpressed in the thymus of transgenic mice relative to nontransgenic mice.

These data provide evidence that transitional B cell maturation can be specifically impaired by the expression of a putative dominant negative form of RAG-I, and raise the possibility that secondary V(D)J rearrangements occur during this window in B cell development. Example 4 Anatomy and Physiology of Transgenic Mice

Transgenic mice were of similar size and weight to their nontransgenic counterparts at two weeks of age. At four weeks, transgenic mice had slightly greater body mass and spleen size. As mice age, the spleen became progressively enlarged. All other organs examined were grossly similar in appearance between transgenic and nontransgenic mice. Preliminary histopathological examination of spleens obtained from a 16 week-old transgenic mouse and its nontransgenic littermate revealed similar splenic architecture. However, immunohistochemical staining with anti-B220 antibody was distinctly weaker in transgenic mice. In normal mice, B220+ B cells were arranged outside the periarteriolar lymphoid sheath. In transgenic mice, fewer cells were strongly stained with anti-B220, and those that were stained darkly were mostly localized proximal to the marginal sinus.

Example 5 B cell functional studies and B cell diversity.

To determine whether the decreased abundance of mature B cells in Tg mice might also be reflected by a reduction in overall serum immunoglobulin levels in Tg mice versus their normal counterparts, serum IgG and IgM levels were quantified in both Tg mice and normal littermates using a commercially available kit (ZeptoMetrix). Interestingly, in Tg mice, levels of serum IgM and IgG are less than half than that observed in normal littermates (FIG. 7). One possible explanation for this observation is that accumulating B220^IowCD19⁺ B cells are non-responsive to antigenic stimulation. To explore this scenario, splenocytes from Tg mice and normal littermates were stimulated in culture for 72h with lipopolysaccharide (LPS), anti-IgM F(ab')2, or control IgG F(ab')2. The metabolic activity of 25,000 cells was then measured using a colorimetric MTT assay described by Mossman (J Immunol Methods 65:55-63 (1983). In this assay, incubation of a pale yellow tetrazolium salt MTT with living cells produces a dark blue formazan product. The amount of substrate converted is dependent on cell viability and activation state. Results show that the splenocytes from Tg mice responded significantly more poorly to stimulation with LPS or anti-IgM cross-linking than splenocytes obtained from non-transgenic littermates (FIG 8). This outcome is consistent with evidence from other laboratories suggesting that transitional B cells are generally non-responsive to antigenic stimulation (Allman et al., J Immunol 167:6834-6840, 2001; Chung et al., Int Immunol 14:157-166, 2002). Given the abundance of B220^IowCD19⁺ B cells in Tg mice, it was possible that they may be derived from a clonal expansion of one or few progenitors, as one might expect in leukemia. To examine this possibility, PCR-based assays of B cell repertoire diversity were performed (Schlissel et al., J Exp Med 173:711-720 (1991). In these assays, D_H-J_H and V_H to D_HJ_H rearrangements are amplified from genomic DNA obtained from spleens of normal and Tg mice by PCR using primers specific for different V, D and J gene families or segments. PCR products are detected by Southern blotting using nested oligonucleotide probes. The results show that the pattern of V(D)J rearrangements in Tg mice wass similar to those seen in normal littermates, except for VHQ52, but the overall abundance of the PCR products is lower in Tg mice (FIGS. 9A-9E). Thus, these results argue against a monoclonal or oligoclonal expansion suggestive of leukemia.

Taken together, the data show that a novel strain of 1 transgenic mice has been developed that exhibits impaired B cell development and the accumulation of an unusual population of B220^lowCD19+ B cells that phenotypically resemble Bl B cells. Compared to normal mice, the transgenic mice appeared to exhibit comparable immune repertoire diversity, but produced less serum immunoglobulin and their splenocytes responded more poorly to proliferative stimuli. Based on these data, catalytically defective RAG-I expression in the transgenic mice may be sufficient to delay, but not completely block, B cell development in the bone marrow. At the immature/Tl B cell stage, endogenous RAG-I expression may be downregulated to a point where the transgene- expressed catalytically defective RAG-I can more completely impair the activity of endogenous RAG-I, particularly if increasing H-2k promoter activity elevates catalytically defective RAG-I levels in the cell (as evidenced by greater H-2kb expression). It is possible that if an immature B cell encounters self-antigen and attempts to alter receptor specificity though receptor editing, it may be blocked from doing so through the action of catalytically defective RAG-I . Of course, since not all immature B cells will exhibit autoreactivity, non-self-reactive B cells should be unaffected by mDDE RAG-I expression and should fully mature. However, those that are induced to undergo receptor editing and cannot might be trapped in a state that is ordinarily transient.

Others recently reported the generation of transgenic mice expressing a DDE mutant of core rabbit RAG-I (residues 380-941) fused at the amino terminus to green fluorescent protein (GFP) driven by the chicken beta-actin (CAG) promoter (Furusawa et al., MoI. Immunol., 39:871-878 (2003)). Of seven founders, only one transmitted the transgene, but no obvious defects in lymphocyte development were detected and no evidence for transgene expression was provided. Founders exhibited reduced serum IgM levels, but serum IgG levels were similar. The difference between the phenotypes observed in the transgenic mice described herein and those described by Furusawa et al. could be attributed to any of the following explanations: (1) attempting to express core rabbit RAG-I in mouse, which may be poorly active in vivo; (2) appendage of a GFP fusion protein to the amino terminus of RAG-I, which my interfere with its function in vivo; or (3) poor transgene expression in lymphocytes, which was untested.

Example 6 Alteration of Receptor Specificity in Response to Self-Reactivity

Receptor editing has been convincingly demonstrated to occur in mice engineered to develop B cells whose antigen receptors recognize class I MHC molecules or dsDNA through the site-directed insertion ("knock-in") of rearranged immunoglobulin light and/or heavy chain variable region exons into endogenous loci (Chen et al., Immunity 3:747-755 (1995), Pelanda et al., Immunity 7:765-775 (1997)). If expression of a catalytically defective RAG-I polypeptide as described herein indeed impairs receptor editing in transgenic mice, then a it is predicted that when the transgene is crossed onto a genetic background prone to receptor editing, the inserted rearranged variable exon would not be susceptible to secondary V(D)J rearrangement events that edit or remove the site-directed transgene DNA sequence. Hence, developing B cells would be expected to retain the antigenic specificity encoded by the "knocked-in" immunoglulin genes.

This example describes a test for whether expression of a catalytically defective RAG-I polypeptide as described herein functionally impairs the initiation of secondary V(D)J rearrangement in a murine model of receptor editing. For these experiments, transgenic mice were bred to mice containing a rearranged site-directed transgene encoding a immunoglobulin heavy chain, called 3H9H/56R, that had been knocked into the heavy chain locus (Li et al., Immunity 15:947-957 (2001)). The sequence of this exon was derived from a hybridoma called 3H9 prepared from a lupus-prone mouse and was subsequently mutated to replace an aspartate with an arginine at position 56 in CDR2. The encoded heavy chain exhibits high affinity and specificity for dsDNA when paired with almost any light chain, with the major exceptions being Vk20, Vk21D, and Vk38c (Li et al., Immunity 15:947-957 (2001)).

Briefly, 3H9H/56R mice were bred to homozygosity using Southern hybridization to confirm the genotype as described (Chen et al., Immunity 3:747- 755 (1995). Next, homozygous male 3H9H/56R mice were bred to female mice hemizygous for the exogenous polynucleotide encoding a catalytically defective RAG-I polypeptide. It was expected that all offspring were hemizygous for the 3H9H/56R knock-in and half the offspring carry the transgenic exogenous polynucleotide (now referred to as mDDE RAG-1/3H9H/56R). Initially, a pair of age-matched hemizygous 3H9H/56R mice, one carrying and the other lacking the mDDE RAG-I transgene were sacrificed at 5 weeks of age (Li et al., Immunity 15:947-957 (2001)), and screened for the presence of 3H9 idiotype-positive and Bl B cells using flow cytometry. To detect the 3H9H/56R idiotype, a monoclonal antibody specific for the 3H9 heavy chain was used, which is produced from hybridoma 1.209 (Obtained from Dr. Weigert, University of Chicago) and conjugated to Alexafluor 647 using a commercially available kit (Molecular Probes). A control cell line expressing the 3H9 heavy chain (provided by Dr. Weigert), was used to verify antibody binding activity after fluorochrome conjugation. Serum obtained from 3H9H/56R and mDDE RAG- 1/3H9H/56R mice was also tested for the presence of anti-dsDNA antibodies using an ELISA as described (Swanson et al,. Biochemistry 35:1624-1633 (1996)). Unexpectedly, one of the mDDE RAG-1/3H9H/56R offspring developed what appeared to be a tumor in or near the eye. Upon dissection, the apparent tumor was found not to be solid, but rather pus-like. Immunophenotypic characterization of the cells in this fluid showed that nearly all the infiltrating cells were granulocytes, as evidenced by their staining with fluorochome- conjugated antibodies specific for CDl Ib (Mac-1) and Gr-I . In principle, if 3H9H/56R+ B cells failed to edit when mDDE RAG-I is expressed, the B cells could produce anti-DNA antibodies if activated. It is tempting to speculate that, due to a scratch or piece of bedding in the eye, an inflammatory response was initiated in mDDE RAG-1/3H9H/56R mice that caused 3H9H/56R+ B cells to become activated, producing anti-DNA antibodies that could form immune complexes with DNA released from dead and dying cells in the initial inflammatory response. The resulting immune complexes could elicit additional inflammation and recruitment of granulocytes, further feeding the response. Alternatively, or in addition, the site could have become infected, and due to underlying immunoglobulin deficiency, mDDE RAG-1/3H9H/56R mice may have failed to control the infection. However, the mouse did not show evident signs of distress, as one might anticipate observing if the animal was suffering from a severe infection. We therefore consider the possibility that the mDDE RAG-1/3H9H/56R mouse may be prone to developing local autoimmune reactions in response to cellular damage. Such a model could be used to develop a better understanding of and treatments for environmentally-induced autoimmune diseases.

In future experiments cohorts of age-matched hemizygous 3H9H/56R mice carrying or lacking the mDDE RAG-I transgene are sacrificed at later ages, for instance, 12 weeks of age (Li et al., Immunity 15:947-957 (2001)), and screened for the presence of 3H9 idiotype-positive and Bl B cells using flow cytometry. Typically, six to eight animals from each group are characterized for these experiments.

Previous studies by others indicate that the 3H9H/56R transgene is edited by two mechanisms. One mechanism involves the physical editing of the 3H9H/56R site-directed transgene by rearrangements that delete or inactivate the targeted allele, resulting in loss of the 3H9H/56R heavy chain and subsequent expression of the heavy chain from the nontargeted allele. The other mechanism involves the functional editing of the 3H9H/56R heavy chain by pairing it with a highly acidic light chain (e.g. Vk21D) which neutralizes the cationic character of 3H9H/56R, thereby reducing the anti-DNA binding activity of the antibody. To obtain these so-called "editor" light chains, multiple rearrangements are generally required, as evidenced by a bias toward Jκ5 rearrangement and the frequent occurrence of CK deletion in 3H9H/56R+ hybridomas studied (Li et al., Immunity 15:947-957 (2001)). If mDDE RAG-I expression in 3H9H/56R mice inhibits receptor editing, a higher number of 3H9H/56R+ B cells should be observed in mDDE RAG-1/3H9H/56R mice compared to their 3H9H/56R counterparts lacking the mDDE RAG-I transgene. However, this outcome may not be observed if editing occurs during a developmental stage when the RAG-I DDE-A transgene is not expressed. For example, as cells transit through the pro- B stage of development, the site-directed 3H9H/56R transgene may become an adventitious target for secondary V(D)J recombination mediated by the RAG complex that is normally expressed during this cell stage to initiate heavy chain gene rearrangement. This form of editing would be considered an antigen- independent process, and might account for the presence of idiotype-negative B cells that could be observed in mDDE RAG-1/3H9H/56R mice. Alternatively, or in addition, B cells in mDDE RAG-1/3H9H/56R mice might fail to edit due to mDDE RAG-I expression, but die as a result, yielding few 3H9H/56R+ positive cells and making it appear that editing is largely unperturbed. If this outcome is observed, hybridomas will be prepared from 3H9H/56R and mDDE RAG- 1/3H9H/56R mice, and clones secreting idiotype-positive and κ-positive immunoglobulin will be examined for JK gene segment usage and evidence of CK deletion on one allele using the PCR assay described by Nemazee and colleagues (Pelanda et al., Immunity 7:765-775 (1997)). In 3H9/56R mice, light chain editors show evidence of a bias toward Jκ5 usage, and exhibit a high frequency of CK deletion. If blocked receptor editing leads to cell death, then the few idiotype- positive B cells that might be observed would likely be derived from B cells in which the initial round of light chain gene rearrangement fortuitously produced a functional light chain editor. In this case, Jκ5 usage should be less biased, and the frequency of CK deletion on one allele among idiotype-positive hybridomas should be lower. If at 12 weeks of age, 3H9/56R mice transgenic for the exogenous polynucleotide described herein exhibit elevated levels of serum anti-DNA antibodies or significantly higher numbers of 3H9 idiotype-positive B cells, another cohort of these mice are assembled and tail bled at various time points thereafter to measure levels of serum anti-DNA antibodies and numbers of 3H9 idiotype-positive B cells. If 3H9/56R mice transgenic for the exogenous polynucleotide described herein begin to show signs of morbidity, mice are sacrificed immediately along with a non-transgenic animal and various organs (especially kidney) are prepared to look for clinical and serological evidence of a lupus-like disease similar to that observed in other murine models of lupus (e.g. [NZB x NZW]Fl and MKL-lpr mice, etc).

The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

AU headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

Claims

What is claimed is:

1. A transgenic mouse comprising in its genome an exogenous polynucleotide, wherein the exogenous polynucleotide comprises a coding sequence encoding a catalytically defective RAG-I polypeptide, or an analog thereof, with an amino acid sequence having at least 80% similarity to SEQ ID NO:2, wherein the mouse exhibits less serum IgG at 4 weeks of age as compared to a wild-type littermate.

2. The transgenic mouse of claim 1 wherein the exogenous polynucleotide comprises a promoter operably linked to the coding sequence, wherein the promoter is a tissue specific promoter that is expressed in lymphoid lineage cells.

3. The transgenic mouse of claim 1 wherein the amino acids of the RAG-I polypeptide, or analog thereof, corresponding to amino acids 600, and 708, and 962 of SEQ ID NO:2 are each independently alanine, glycine, serine, threonine, or proline.

4. The transgenic mouse of claim 1 wherein the amount of serum IgG exhibited by the mouse at 4 weeks of age is reduced 3-fold as compared to a wild-type littermate.

5. A cell obtained from the transgenic mouse of claim 1, wherein the cell comprises the exogenous polynucleotide.

6. The transgenic mouse of claim 1 wherein the mouse is chimeric for the exogenous polynucleotide.

7. The transgenic mouse of claim 1 wherein the mouse is heterozygous for the exogenous polynucleotide.

8. A transgenic mouse comprising in its genome an exogenous polynucleotide, wherein the exogenous polynucleotide comprises a nucleotide sequence having at least 80% similarity to SEQ ID NO: 1 and encoding a catalytically defective RAG-I polypeptide, or an analog thereof, wherein the mouse exhibits less serum IgG at 4 weeks of age as compared to a wild-type littermate.

9. The transgenic mouse of claim 8 wherein the exogenous polynucleotide comprises a promoter operably linked to the coding sequence, wherein the promoter is a tissue specific promoter that is expressed in lymphoid lineage cells.

10. The transgenic mouse of claim 8 wherein the amount of serum IgG exhibited by the mouse at 4 weeks of age is reduced 3-fold as compared to a wild-type littermate.

11. A cell obtained from the transgenic mouse of claim 8, wherein the cell comprises the exogenous polynucleotide.

12. The transgenic mouse of claim 8 wherein the mouse is chimeric for the exogenous polynucleotide.

13. The transgenic mouse of claim 8 wherein the mouse is heterozygous for the exogenous polynucleotide.

14. A method for making a transgenic mouse comprising: introducing into a fertilized mouse egg an exogenous polynucleotide comprising a coding sequence encoding a catalytically defective RAG-I polypeptide, or an analog thereof, with an amino acid sequence having at least 80% similarity to SEQ ID NO:2; implanting in a female mouse the fertilized mouse egg comprising the exogenous polynucleotide to produce a chimeric mouse, wherein the chimeric mouse comprises in a germ cell the exogenous polynucleotide.

15. A transgenic mouse comprising cells expressing a catalytically defective RAG-I polypeptide, or an analog thereof, with an amino acid sequence having at least 80% similarity to SEQ ID NO:2, wherein the mouse exhibits less serum IgG at 4 weeks of age as compared to a wild-type littermate.

16. The transgenic mouse of claim 15 wherein the amino acids of the RAG-I polypeptide, or analog thereof, corresponding to amino acids 600, and 708, and 962 of SEQ ID NO:2 are each independently alanine, glycine, serine, threonine, or proline.

17. The transgenic mouse of claim 15 wherein the amount of serum IgG exhibited by the mouse at 4 weeks of age is reduced 3-fold as compared to a wild-type littermate.

18. A cell obtained from the transgenic mouse of claim 15, wherein the cell comprises the exogenous polynucleotide.

19. The transgenic mouse of claim 15 wherein the mouse is chimeric for the exogenous polynucleotide.

20. The transgenic mouse of claim 15 wherein the mouse is heterozygous for the exogenous polynucleotide.