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WO2006110309A2 - Souris transgeniques exprimant une population unique de lymphocytes b et methodes d'utilisation - Google Patents

Souris transgeniques exprimant une population unique de lymphocytes b et methodes d'utilisation Download PDF

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WO2006110309A2
WO2006110309A2 PCT/US2006/011105 US2006011105W WO2006110309A2 WO 2006110309 A2 WO2006110309 A2 WO 2006110309A2 US 2006011105 W US2006011105 W US 2006011105W WO 2006110309 A2 WO2006110309 A2 WO 2006110309A2
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rag
mouse
exogenous polynucleotide
cells
polypeptide
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WO2006110309A3 (fr
WO2006110309A9 (fr
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Patrick Swanson
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Creighton University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0381—Animal model for diseases of the hematopoietic system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

  • 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.
  • V(D)J recombination 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).
  • DSBs DNA double strand breaks
  • NHEJ non-homologous end joining pathway
  • RAG-mediated cleavage generates two distinct DNA ends: blunt, 5' ⁇ hosphorylated 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)).
  • 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)).
  • 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.
  • the rearranged light chain gene is functionally tested by pairing the expressed light chain with the ⁇ 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.
  • slgD begin to appear through alternative splicing of the heavy chain RNA transcript.
  • 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)).
  • this "receptor editing" process involves either the replacement of the offending light chain variable ex on or kappa deletion to promote ⁇ light chain rearrangement (Gay et al., J.
  • 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.
  • PALS periarteriolar lymphoid sheath
  • IgD begins to appear on the cell surface (slgD) through alternative splicing of the heavy chain RNA transcript.
  • the T2 B cell migrates to the lymphoid follicle surrounding the PALS.
  • Significant functional differences exist in the response of Tl and T2 B cells to antigenic stimulation.
  • 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.
  • MZ B cells 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.
  • CD21 complement receptor 2
  • 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 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG 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).
  • B22O 10 and B220 hi 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 CD19 and B220, and B220 10 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.
  • FIG. 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).
  • 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.
  • the present invention includes a transgenic animal, for instance, a transgenic mouse, having in its genome an exogenous polynucleotide.
  • 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.
  • the exogenous polynucleotide also includes one or more regulatory regions operably linked to the coding region.
  • polypeptide refers broadly to a polymer of two or more amino acids joined together by peptide bonds.
  • 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).
  • peptide oligopeptide
  • 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.
  • present invention also includes the exogenous polynucleotide, and the polypeptide it encodes.
  • 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.
  • 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) lo CD19 + , 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.
  • 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)).
  • 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)).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • conservative substitutions which are selected from other members of the class to which the amino acid belongs.
  • an amino acid belonging to a grouping of amino acids having a particular size or characteristic can generally be substituted for another amino acid without substantially altering the structure of a polypeptide.
  • 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 IY: His, Arg and Lys (representing basic side chains); Class V: He, VaI, Leu, Phe and Met
  • 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.
  • 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.
  • 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.
  • identity structural similarity
  • 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 MgCl 2 or MnCl 2 .
  • 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.
  • 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 MgCl 2 (or MnCl 2 ) is replaced with CaCl 2 . 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) lo CD19 + 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) lo CD19 + , 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.
  • “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 55 0 C.
  • 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.
  • 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.
  • 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.
  • two nucleotide sequences are compared using the BESTFIT algorithm in the GCG package (version 10.2, Madison WI), or the Blastn program of the BLAST 2 search algorithm, as described by Tatusova, et al.
  • 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 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.
  • 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.
  • 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.
  • promoters 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.
  • a promoter including a tissue specific promoter, can include a portion of an exon.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • a vector is capable of replication in a bacterial host, for instance E. coli, a eukaryotic host such as a mouse cell, or both.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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 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 hi IgD l0 CD21 lo CD23 /lo CD24 int CD44 hi CD62U /Io AA4. rCD5 l0 H2Kb 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.
  • 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 10 2 -10 3 absorbance units. For comparison, the level of B220 expression in the "B220 10 " B cells in transgenic mice is between lO'-lO 2 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.
  • 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.
  • B-CLL B- chronic lymphocytic leukemia
  • 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.
  • B-CLL a fairly common form of leukemia
  • B-CLL 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%).
  • 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.
  • 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)).
  • the transgenic animals described herein may serve as a useful model for progressive hypogammaglobulinemia associated with an accumulating Bl -like B cell population.
  • 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), D ⁇ OOARev:
  • 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).
  • 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 ⁇ -BstZ ⁇ ll 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 BamH ⁇ 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.
  • 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'- TGGTCGACTCT AGAGGATCCAC-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'.
  • 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.
  • 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.
  • 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 BsrG ⁇ restriction fragment encoding residues 484-727 to detect BamHl -digested genomic DNA (see FIG. 2).
  • the construct-specific primers were H2kb-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.
  • Tg expression may be assessed both at the transcriptional level and at the protein level, as they are not necessarily directly correlated.
  • 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.
  • 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.
  • 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).
  • primary lymphoid organs e.g. bone marrow and thymus
  • secondary lymphoid organs e.g. spleen and lymph node.
  • 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.
  • 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 ⁇ -actin expression using the comparative threshold cycle (C 1 ) 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).
  • 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).
  • Flow cytometry is the method of choice for analyzing lymphocyte development.
  • 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; Converse 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).
  • 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.
  • B220 lo CD19 + B cells were slightly larger and more granular than B220 hi CD19 + B cells from normal mice, as evidenced by their higher mean forward and side scatter properties, and were characterized as being IgM h TgD lo CD21 l0 CD23 " /l0 CD24 int CD44 hi CD62L "/l0 AA4.r (Fig. 6B). These cells expressed low levels of CD5, comparable to peritoneal B22O 10 B cells (Fig. 6C), and CD43, but did not express the germinal center marker GL-7.
  • the B220 lo CD19 + B cells resembled Tl, MZ and Bl B cells with respect to surface IgM and IgD expression.
  • B220 lo CD19 + B bear little resemblance to Tl and MZ B cells. Instead, since B220 lo CDl 9 + B cells share the forward and side scatter properties of B 1 B cells, and display a similar staining pattern with antibodies to CD5 and B220, it was tentatively concluded that the B220 lo CD19 + B cells observed in the transgenic mice most closely resembled the Bl B cell. At 4 weeks of age, the splenic B220 hl CD19 + 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.
  • IgM + IgD " immature
  • 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.
  • 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.
  • LPS lipopolysaccharide
  • 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.
  • PCR-based assays of B cell repertoire diversity were performed (Schlissel et al., J Exp Med 173:711-720 (1991).
  • D H -J H and V H to D H J 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).
  • 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- 1 , 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 .
  • non-self-reactive B cells should be unaffected by mDDE RAG-I expression and should fully mature.
  • 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
  • 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.
  • 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)).
  • 3H9H/56R mice were bred to homozygosity using Southern hybridization to confirm the genotype as described (Chen et al., Immunity 3:747- 755 (1995).
  • homozygous male 3H9H/56R mice were bred to female mice hemizygous for the exogenous polynucleotide encoding a catalytically defective RAG-I polypeptide.
  • 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)).
  • 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.
  • mice did not show evident signs of distress, as one might anticipate observing if the animal was suffering from a severe infection.
  • Such a model could be used to develop a better understanding of and treatments for environmentally-induced autoimmune diseases.
  • 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.
  • a highly acidic light chain e.g. Vk21D
  • 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 ⁇ -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)).
  • 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.
  • 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).

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Abstract

La présente invention porte sur des animaux transgéniques, tels que des souris transgéniques, qui expriment une forme dominante-négative de RAG-I dans la phase de transition du développement des lymphocytes B, ce qui permet un réarrangement initial génétique du récepteur à l'antigène, mais bloque l'édition du récepteur et/ou la révision du récepteur qui se produit pendant les dernières phases du développement. Selon un aspect de cette invention, l'animal transgénique comprend dans son génome un polynucléotide exogène comprenant une séquence codante codant un polypeptide RAG-I catalytiquement défectueux ou un analogue de celui-ci. L'invention porte également sur des méthodes de fabrication et d'utilisation de ces animaux transgéniques.
PCT/US2006/011105 2005-03-26 2006-03-27 Souris transgeniques exprimant une population unique de lymphocytes b et methodes d'utilisation WO2006110309A2 (fr)

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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
FURUSAWA T ET AL: "Catalytic RAG1 mutants obstruct V(D)J recombination in vitro and in vivo." MOLECULAR IMMUNOLOGY, vol. 39, no. 14, May 2003 (2003-05), pages 871-878, XP002407203 ISSN: 0161-5890 cited in the application *
KIM D R ET AL: "Mutations of acidic residues in RAG1 define the active site of the V(D)J recombinase" GENES AND DEVELOPMENT, vol. 13, no. 23, December 1999 (1999-12), pages 3070-3080, XP002407205 ISSN: 0890-9369 cited in the application *
LANDREE M A ET AL: "Mutational analysis of RAG1 and RAG2 identifies three catalytic amino acids in RAG1 critical for both cleavage steps of V(D)J recombination" GENES AND DEVELOPMENT, vol. 13, no. 23, December 1999 (1999-12), pages 3059-3069, XP002407204 ISSN: 0890-9369 cited in the application *
PARKS K W ET AL: "Transgenic mice expressing catalytically inactive RAG-1 exhibit hypogammaglobulinemia and altered B cell development, but not autoreactivity" JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY, vol. 117, no. 2, Suppl. S, February 2006 (2006-02), page S100, XP002407202 & 62ND ANNUAL MEETING OF THE AMERICAN-ACADEMY-OF-ALLERGY-ASTHMA-AND-IMM UNOLOGY; MIAMI BEACH, FL, USA; MARCH 03 -07, 2006 ISSN: 0091-6749 *

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