WO1995014377A2 - Modele pour maladies infectieuses - Google Patents

Modele pour maladies infectieuses Download PDF

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Publication number
WO1995014377A2
WO1995014377A2 PCT/IB1994/000417 IB9400417W WO9514377A2 WO 1995014377 A2 WO1995014377 A2 WO 1995014377A2 IB 9400417 W IB9400417 W IB 9400417W WO 9514377 A2 WO9514377 A2 WO 9514377A2
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WO
WIPO (PCT)
Prior art keywords
mammal
transgene
cells
knockout
promoter
Prior art date
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PCT/IB1994/000417
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English (en)
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WO1995014377A3 (fr
Inventor
Rae Suk-Man Yeung
Tak W. Mak
Original Assignee
Amgen Canada Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Amgen Canada Inc. filed Critical Amgen Canada Inc.
Priority to AU10758/95A priority Critical patent/AU1075895A/en
Priority to EP95901576A priority patent/EP0734201A1/fr
Priority to JP7514946A priority patent/JPH09500025A/ja
Publication of WO1995014377A2 publication Critical patent/WO1995014377A2/fr
Publication of WO1995014377A3 publication Critical patent/WO1995014377A3/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027—New breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0278—Humanized animals, e.g. knockin
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027—New breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knockout animals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • 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; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; 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; CARE OF BIRDS, FISHES, INSECTS; 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
    • A01K2267/0337—Animal models for infectious diseases
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; 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

Definitions

  • This invention relates to a non-human mammal produced by recombinant DNA technology. More specifically, the invention concerns a non-human mammal that is susceptible to sepsis and other diseases, and that is useful for screening potential therapeutic compounds.
  • the in vivo system used for data collection would be a human being; however, for ethical and pragmatic reasons, laboratory animals, and not human beings, are typically used as in vivo screening systems for drug development.
  • laboratory animals can be helpful for use as model systems to assess the efficacy of a potential therapeutic and to identify the possible side- effects of the therapeutic
  • models have limitations.
  • One significant limitation concerns the differences between laboratory animals and humans in disease resistance and susceptibility. These differences are often due to inter-species differences in the structure and function of the immune system. Therefore, to evaluate a chemical compound for its therapeutic value in humans, it would be useful to have an animal model with an immune system that resembles the human immune system.
  • transgenic mammal generation and knockout mammal generation have been employed by researchers to produce animals that either do not express an endogenous gene (knockout animals) , or to produce animals that contain one or more exogenous or heterologous genes (transgenic animals) .
  • transgenic mammals involves the insertion of a novel nucleic acid sequence, often called a transgene, into one or more chromosomes of the mammal.
  • the transgene typically comprised of DNA, codes for a particular polypeptide.
  • the transgene is typically inserted via microinjection into the pronucleus of an egg where it is incorporated into the DNA of the developing embryo. This embryo is then implanted into a "surrogate host" for the duration of gestation. The offspring of the surrogate host are evaluated for the presence of the novel DNA.
  • transgene i . e .
  • production of the protein encoded by the transgene DNA sequence may confer a new phenotype on the mammal.
  • control elements such as promoter and enhancer/silencer
  • the mammal may become more or less susceptible to a particular disease or series of diseases.
  • Such transgenic mammals are valuable for screening and testing compounds that may be useful in treating or preventing the disease (s), and/or for developing methods useful in the treatment or diagnosis of the disease.
  • a knockout mammal requires insertion of a nucleic acid sequence (usually DNA) that is designed to suppress expression of the gene to be "knocked out" into an undifferentiated cell line termed an embryonic stem cell, or ES cell.
  • ES cell an embryonic stem cell
  • the ES cell is injected into a developing mammalian embryo where it hopefully will be integrated into, and become part of, the embryo during development. The embryo is then implanted into a surrogate host for the duration of gestation.
  • the immune system of every mammalian species is comprised of many specialized cells that act together in a highly complex manner to protect the mammal from dangerous foreign substances such as a variety of pathogens, toxins, and the like.
  • the MHC major histocompatibility complex
  • the mouse MHC is known as the H-2 complex (“H-2")
  • the human MHC is referred to as the HLA complex (“HLA”) .
  • the HLA complex is comprised of more than 100 genes (Klein et al . , Sci . Am. , 269:78-83 [1993]) .
  • MHC MHC protein
  • MHC class I proteins are membrane bound proteins that are expressed on the surface of almost all nucleated cells; MHC class II proteins
  • ⁇ II proteins are membrane bound proteins that are expressed only on the surface of certain types of immune system cells, namely dendritic cells, macrophages, and B cells.
  • the MHC class II proteins play a critical role in the immune system's recognition and attack of foreign substances.
  • APCs antigen presenting cells
  • macrophages which bind to the substance, ingest it and process it.
  • the processed portions of the substance may be attached to MHC class II proteins on the surface of the macrophage.
  • This presentation of "display" of the substance bound to the MHC on the surface of the macrophage enables other cells of the immune system, such as certain T cells, to recognize and respond to the foreign substance.
  • T cells become “activated” and are induced to proliferate and to release compounds that are harmful to the foreign substance.
  • activation of T cells serves to activate other cells of the immune system to also mount an attack on the substance.
  • T cells one type of immune system cell, express a variety of proteins on their cell surface. Many of these proteins are involved in recognition and binding of extracellular substances, and/or in cell signaling. Two important T cell surface molecules are
  • CD4 and CD8 are glycoprotein monomer of molecular weight about 55kD.
  • the murine CD4 is encoded by L3T4 and is about 26 kb in size.
  • CD8 is a disulfide-linked heterodimer.
  • the genes encoding the two CD8 monomers are referred to as CD8 alpha and CD8 beta.
  • the monomers comprising the CD8 heterodimer are encoded by the genes Lyt-2 and Lyt-3. Lyt-2 is about 4.4 kb in size and contains 5 exons.
  • Immature T cells express both CD4 and CD8 on their cell surface. As the cells mature, they cease to express either CD4 or CD8. Thus, mature T cells are classified based on whether they express CD4 or CD8. Generally, mature T cells that express only CD4 are called T helper cells, and recognize and respond to antigens bound to MHC class II molecules. Generally, mature T cells that express only CD8 are called cytotoxic T cells, and recognize and respond to antigens bound to MHC class I molecules.
  • MHC, CD4, and CD8 molecules differ in their amino acid sequence between species. Thus, one species may recognize a pathogen or substance as foreign and mount a response to it, while another species may not. In addition, there are intra-species differences in the response to any one toxin or pathogen (i.e., different species and even members of the same species may be extremely sensitive to a particular toxin or pathogen, while others are more tolerant of its presence in the body) . This is due to the many different allelic variations of the MHC loci, (as discussed above) . Each individual member of a species expresses only one or at most two of these allelic forms at each MHC locus, and different allelic forms differ in their ability to present or bind different pathogens.
  • mice carrying the transgenes encoding the alpha and beta chains of the human HLA-DQw6 protein, a member of the MHC class II protein family.
  • Barzaga-Gilbert et al . J. Exp . Med. , 175:1707-1715 [1992]) describe a mouse containing a transgene encoding human CD4.
  • Killeen et al . (EMBO J. , 12:1547-1553 [1993]) describe a mouse lacking expression of endogenous CD4 but containing a transgene encoding human CD4.
  • Rahemtulla et al . (Nature, 353:180-184 [1991]) describe a mouse that does not express endogenous CD4.
  • Fung-Leung et al . (Cell, 65:443-449 [1991]) describe a mouse that lacks expression of the endogenous CD8 molecule.
  • sepsis The presence of disease causing organisms and/or their toxins in human blood or tissues can result in a condition known as sepsis.
  • the symptoms of sepsis induced by such organisms as E coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter aerogenes, and Neisseria meningitidis can include fever, diarrhea, a drop in blood pressure, leaky blood vessels, and/or disseminated blood clotting in a variety of organs. It has been estimated that the mortality rate from sepsis is about 35 percent (Aldridge, TIBTech, 11:373-375 [1993]), and up to 50 percent in septic shock.
  • septic shock One severe form of sepsis is termed septic shock and results when an individual with sepsis experiences a large drop in blood pressure.
  • Treatment for septic shock requires immediate restoration of blood pressure which is typically accomplished by administering fluids and/or inotropic agents, and broad spectrum antibiotics.
  • toxins that have been identified as the causative agents in sepsis. Among these are exotoxins (toxins secreted by the invading organism) and endotoxins (toxins that are a component of the cell wall of the invading organism, e . g. , LPS, or lipopolysaccharide) .
  • enterotoxins proteins that are made by various species of both Streptococci and Staphylococcus, and share a similar mechanism of action in activating the body's immune system to mount an attack on the pathogen.
  • enterotoxins are known as "superantigens" .
  • the mechanism of superantigen binding to MHCII and T cells is distinct from that of other antigens.
  • Superantigens bind simultaneously to the T cell receptor and the MHCII molecule, thereby activating a large number of T cells irrespective of the antigen binding specificity of the T cell. This results in the release of large amounts of cytokines in the body, which ultimately leads to shock.
  • the mechanism of binding for conventional antigens is to bind to a very well defined groove in a specific MHCII molecule; this antigen/MHCII complex is then recognized only by a highly specific subset of T cells, i . e . , those that express a receptor on their surface that is specific for the particular antigen/MHCII complex (1 in IO 7 T cells) .
  • T cells i . e . , those that express a receptor on their surface that is specific for the particular antigen/MHCII complex (1 in IO 7 T cells) .
  • fewer T cells are activated by a normal antigen binding as compared with superantigen responses, which trigger up to 40 percent of the total T cell population.
  • antibiotic therapy is often used to treat sepsis, it is not always effective, and can have a number of detrimental side effects.
  • steroids may also be indicated to modulate the activity of the immune system.
  • blood products such as fresh frozen plasma or anticoagulants such as heparin may be used to counter the effects of the immune system's complement cascade activity which leads to clotting.
  • the present invention provides a non-human mammal or its progeny lacking expression of endogenous CD4 and CD8, wherein the mammal has inserted a nucleotide sequence comprising the DNA encoding human CD4 or a biologically active fragment thereof and a nucleotide sequence comprising the DNA encoding an allele of the HLA DQ locus or a biologically active fragment thereof.
  • the invention provides a process for preparing a mammal or its progeny comprising suppressing expression of the nucleotide sequence encoding endogenous CD8 in the mammal; suppressing expression of the nucleotide sequence encoding endogenous CD4 in the mammal; inserting nucleotide sequences for an allele of the HLA DQ locus alpha and beta chains, or biologically active fragments thereof into the mammal; and inserting a nucleotide sequence for human CD4 or a biologically active fragment thereof into the mammal.
  • the invention provides a method of screening a compound for its anti-sepsis effect, comprising exposing a mammal lacking expression of endogenous CD4 and CD8, wherein the mammal has inserted a nucleotide sequence comprising the DNA encoding human CD4 or a biologically active fragment thereof and a nucleotide sequence comprising the DNA encoding an allele of the HLA DQ locus or a biologically active fragment thereof to a compound or organism that has the capacity to induce sepsis administering to the mammal a therapeutically effective amount of the compound and screening the mammal for sepsis or sepsis ⁇ like symptoms.
  • Figure 1 depicts the knockout construct used to suppress expression of Lyt-2, the mouse CD8 gene (mCD8-/-) .
  • the dark boxes represent exons of Lyt-2 (indicated by Roman numerals) with the shaded areas denoting coding regions.
  • the neomycin phosphotransferase gene is indicated by the hatching. Selected restriction enzymes are indicated.
  • Figure 2 depicts the knockout construct used to suppress expression of the mouse CD4 gene (mCD4-/-) .
  • the neomycin phosphotransferase gene is indicated by the hatching.
  • the dark boxes represent exons of the mCD4 gene (indicated by Roman numerals) . Selected restriction enzymes are indicated.
  • Figure 3 depicts the transgene construct used to generate human CD4 (hCD4) transgenic mice.
  • the hCD2 5' promoter region and the hCD2 3' regulatory region are indicated as are the hCD4 cDNA insert and the hCD2 minigene consisting of the hCD2 exon I genomic sequence and the hCD2 cDNA coding region for the remainder of the gene. Selected restriction enzymes are also indicated.
  • Figure 4 depicts the breeding scheme used to generate a double knockout, double transgenic mouse.
  • hCD4 represents a human CD4 transgenic mouse
  • DQw6 represents a human DQw ⁇ (alpha and beta chains) transgenic mouse
  • mCD4 represents the mouse endogenous CD4
  • mCD8 represents the mouse endogenous CD8 (Lyt-2 gene) .
  • the symbol “hCD4+/-” refers to a mouse heterozygous for the hCD4 transgene
  • the symbols "mCD4/8-/-” and “mCD4/8+/-” refer to a mouse that is a heterozygote knockout of CD4 and CD8.
  • Figure 5 depicts a dose-response curve measuring T cell response or proliferation (indicated as 3H thymidine uptake) when stimulated by decreasing amounts of SEB in freshly killed ex vivo cells of different mice genotypes.
  • concentration of SEB is indicated on the X axis.
  • Dark circles represent the double knockout, double transgenic mouse (mCD4-/-, mCD8- /-, DQw6+, hCD4+)
  • open circles represent the double knockout (mCD4-/-, mCD8-/-) with the hCD4 transgene.
  • dark squares represent C57BL/6 wild type, and the open pentagonal represents double knockout mice.
  • knockout refers to partial or complete reduction of the expression of at least a portion of a polypeptide encoded by an endogenous DNA sequence in a single cell, selected cells, or all of the cells of a mammal.
  • knockout construct refers to a nucleotide sequence that is designed to decrease or suppress expression of a polypeptide encoded by endogenous DNA sequences in a cell.
  • the nucleotide sequence used as the knockout construct is typically comprised of (1) DNA from some portion of the endogenous gene (exon sequence, intron sequence, and/or promoter sequence) to be suppressed and (2) a marker sequence used to detect the presence of the knockout construct in the cell.
  • the knockout construct is inserted into a cell, and integrates with the genomic DNA of the cell in such a position so as to prevent or interrupt transcription of the native DNA sequence.
  • the knockout construct nucleotide sequence may comprise 1) a full or partial sequence of one or more exons and/or introns of the gene to be suppressed, 2) a full or partial promoter sequence of the gene to be suppressed, or 3) combinations thereof.
  • the knockout construct is inserted into an embryonic stem cell (ES cell) where the cell is an undifferentiated cell, usually derived from an embryo of the same species as the developing embryo into which it is subsequently injected and is integrated into the ES cell genomic DNA, usually by the process of homologous recombination.
  • ES cell embryonic stem cell
  • This ES cell is then injected into, and integrates with, the developing embryo.
  • disrupting of the gene refers to insertion of a nucleotide sequence into one region of an endogenous gene (usually one or more exons) and/or the promoter region of a gene so as to decrease or prevent expression of that gene in the cell. Insertion is usually accomplished by homologous recombination.
  • gene refers to the sequence of nucleotides or codons encoding a polypeptide or fragment of a polypeptide.
  • a nucleotide sequence knockout construct can be prepared by inserting a nucleotide sequence comprising an antibiotic resistance gene into a portion of an isolated nucleotide sequence that is complementary to the endogenous DNA sequence (promoter and/or coding region) to be disrupted.
  • the construct will typically integrate into the genomic DNA.
  • many progeny of the cell will no longer express the gene at least in some cells, or will express it at a decreased level, as the nucleotide sequence of the gene is now disrupted by the antibiotic resistance gene.
  • marker sequence refers to a nucleotide sequence that is (1) used as part of a larger nucleotide sequence construct (i.e., the "knockout construct") to disrupt the expression of the gene(s) of interest (such as, for example, CD4 and/or CD8) , and (2) used as a means to identify those cells that have incorporated the knockout construct into the genome.
  • the marker sequence may be any sequence that serves these purposes, although typically it will be a sequence encoding a protein that confers a detectable trait on the cell, such as an antibiotic resistance gene or an assayable enzyme not typically found in the cell. Where the marker sequence encodes a protein, the marker sequence will also typically contain either a homologous or heterologous promoter that regulates its expression.
  • transgene refers to an isolated nucleotide sequence that may or may not be operably linked to a promoter, and that is inserted into one or more cells of a mammal or mammalian embryo.
  • the transgene may be comprised of a nucleotide sequence that is either homologous or heterologous to a particular nucleotide sequence in the mammal's endogenous genetic material, or that is a hybrid sequence (i.e. one or more portions of the transgene are homologous, and one or more portions are heterologous to the mammal's genetic material) .
  • the transgene nucleotide sequence may encode a polypeptide or a variant of a polypeptide, found endogenously in the mammal, it may encode a polypeptide not naturally occurring in the mammal ( i . e . an exogenous polypeptide) , or it may encode a hybrid of endogenous and exogenous polypeptides.
  • the transgene is operably linked to a promoter
  • the promoter may be homologous or heterologous to the mammal and/or to the transgene.
  • the promoter may be a hybrid of endogenous and exogenous promoter elements (enhancers, silencers, suppressors, and the like) .
  • CD4 refers to a cell surface glycoprotein expressed primarily on T helper cells of mammalian species, and is believed to be involved in cell signaling primarily via recognition and binding as a co-receptor to MHCII molecules that have bound antigen and are recognized by the T cell receptor.
  • human CD4 may have the wild-type nucleotide sequence, or it may be an insertional, deletional, and/or substitutional variant thereof.
  • mouse CD4 may have the wild type nucleotide sequence, or it may be an insertional, deletional, and/or substitutional variant thereof.
  • insertional, deletional and/or substitutional variant includes, inter alia, allelic variants.
  • CD8 refers to a cell surface glycoprotein that is typically comprised in mammalian species of two heterologous monomer polypeptides. CD8 is expressed primarily on the surface of cytotoxic T cells, and is believed to be involved in cell signaling primarily via recognition and binding as a co-receptor to MHCI molecules that have bound antigen and are recognized by the T cell receptor.
  • the human CD8 monomers may have the wild type alpha and beta chain nucleotide sequences, or may be insertional, deletional, and/or substitutional variants thereof, including, inter alia, allelic variants.
  • Mouse CD8 monomers may have the wild type nucleotide sequences of the Lyt-2 and Lyt-3 genes, or may be insertional, deletional, and or substitutional variants thereof, including, inter alia, allelic variants.
  • the term "allele of the HLA DQ locus" refers to any and all variants of the human major histocompatibility complex class II (MHCII) DQ locus, and includes all polypeptides, or monomers of that locus (i . e . , alpha and beta chains) , which are combined to generate the final protein known as the product of the DQ locus.
  • the DQw6 allele is comprised of alpha and beta monomers, and the monomers together comprise one allelic product of the DQ locus.
  • operably linked refers to the arrangement of various nucleotide sequences relative to each other such that the elements are functionally connected and are able to interact with each other.
  • Such elements may include, without limitation, one or more promoters, enhancers, polyadenylation sequences, and transgenes.
  • the nucleotide sequence elements when properly oriented, or operably linked, act together to modulate the activity of one another, and ultimately may affect the level of expression of the transgene (s) . By modulate is meant increasing, decreasing, or maintaining the level of activity of a particular element.
  • the position of each element relative to other elements may be expressed in terms of the 5' terminus and the 3' terminus of each element, and the distance between any particular elements may be referenced by the number of intervening nucleotides, or base pairs, between the elements.
  • biologically active fragment refers to a nucleotide sequence that is less than the full-length genomic or cDNA nucleotide sequence of a gene or transgene, but contains a sufficient portion of the full length nucleotide sequence such that the gene (or transgene) product of the biologically active fragment possesses at least a portion of the biological activity possessed by the gene product of the full length sequence.
  • rodent and “rodents” refer to all members of the phylogenetic order -Rodentia including any and all progeny of all future generations derived therefrom.
  • murine refers to any and all members of the family Muridae, including rats and mice.
  • progeny refers to any and all future generations derived and descending from a particular mammal, i . e . , a mammal containing a knockout construct inserted into its genomic DNA. Thus, progeny of any successive generation are included herein such that the progeny, the FI, F2, F3, generations and so on indefinitely are included in this definition.
  • mammal refers to all members of the class Mammalia except humans and includes progeny of the mammal.
  • immunomodulate and “immunomodulation” refer to changes in the level of activity of any components of the immune system as compared to the average activity of that component for a particular species. Thus, as used herein, immunomodulation refers to an increase or a decrease in activity. Immunomodulation may be detected by assaying the level or function of B cells, any or all types of T cells, antigen presenting cells, and any other cells believed to be involved in immune function.
  • immunomodulation may be detected by evaluating 1) the level of expression of particular genes believed to have a role in the immune system, 2) the level of particular compounds such as cytokines (interleukins and the like) or other molecules that have a role in the immune system, and/or 3) the level of particular enzymes, proteins, and the like that are involved in immune system functioning.
  • tissue damage refers to the presence of disease causing organisms and/or their toxins in the blood and/or tissues of a mammal.
  • anti-sepsis effect refers to a situation wherein the symptoms of sepsis or the sepsis condition are reduced or eliminated.
  • therapeutic regimen refers to a treatment designed to achieve a particular effect i.e., reduction or elimination of a detrimental condition or disease.
  • the treatment may include administration of one or more compounds either simultaneously or at different times, for the same or different amounts of time. Alternatively, or additionally, the treatment may include exposure to other therapies such as radiation, a particular diet, physical therapy, and the like.
  • This invention contemplates a mammal in which at least two genes of the immune system have been disrupted or knocked out. Included within the scope of this invention however, is a mammal with more than two genes knocked out.
  • the gene(s) to be knocked out or disrupted are selected from CD4 and CD8. At least some sequence information on the genes to be disrupted must be available for preparation of both the knockout construct and the screening probes.
  • the sequence information may be from a species other than the species to be genetically manipulated by insertion of the knockout construct, provided that it is reasonably believed that the nucleotide sequences from each species will be substantially homologous.
  • the nucleotide sequence comprising the knockout construct will be comprised of one or more exon and/or intron regions from the genomic DNA sequence, and/or a promoter region.
  • the DNA to be used may alternatively be a cDNA sequence provided that the cDNA is sufficiently large.
  • the DNA used in the knockout construct will be at least about 1 kilobase (kb) in length and preferably 3-4 kb in length, thereby providing sufficient complementary sequence for homologous recombination or hybridization to genomic DNA when the knockout construct is introduced into ES cell (discussed below) .
  • kb kilobase
  • the DNA used in the knockout construct will be at least about 1 kilobase (kb) in length and preferably 3-4 kb in length, thereby providing sufficient complementary sequence for homologous recombination or hybridization to genomic DNA when the knockout construct is introduced into ES cell (discussed below) .
  • kb kilobase
  • 3-4 kb in length
  • mice not expressing either copy of gene A i.e., gene A knockout; homozygous
  • mice not expressing either copy of gene B i.e., gene B knockout; homozygous
  • offspring "F ⁇ ' that are heterozygous for both mutations (i.e., have one of two copies of both mutant genes [A+/-, B+/-] ) .
  • the offspring (Fi) can then be mated, and if the genes segregate separately (in Mendelian fashion) then one- sixteenth of the offspring (F2) will be homozygous for both mutations and thus will be double knockouts (i.e., A-/-, B-/-) . Mice with these genotypes may be screened by suitable assays to identify the absence of expression of each gene product.
  • nucleotide sequence (s) comprising the knockout construct (s) can be obtained using methods well known in the art such as those described by Sambrook et al .
  • Such methods include, for example, screening a genomic library with a cDNA probe that has a suitable level of homology to the genomic sequence such that the genomic clone can be identified.
  • the cDNA may be obtained by screening a cDNA library with oligonucleotide probes or antibodies (where the library is cloned into an expression vector) .
  • a promoter sequence is to be used in the knockout construct
  • synthetic DNA probes can be designed for screening a genomic library containing the promoter sequence, or a suitably homologous promoter may be used as a screening probe.
  • Another method for obtaining the nucleotide sequence comprising the knockout construct is to manufacture this sequence synthetically, using methods such as those described in Engels et al . (Agnew Chem. Int . Ed. Eng. ) 28:716-734 [1989] . These methods include, inter alia, the phosphotriester, phosphoramidite, and H-phosphonate methods of nucleic acid synthesis.
  • the nucleotide sequence comprising the knockout construct must be generated in sufficient quantity for later genetic manipulation.
  • Amplification of the nucleotide sequence to achieve the desired quantity may be conducted by 1) placing the sequence into a suitable vector and transforming bacterial or other cells that can rapidly amplify the vector, 2) by PCR amplification, or 3) by synthesis using methods set forth by Engels et ai, supra .
  • the nucleotide sequence comprising the knockout construct is typically digested with one or more restriction enzymes selected to cut at a location(s) such that a new DNA sequence encoding a marker gene can be inserted in the proper position within this nucleotide sequence.
  • the proper position for marker gene insertion is that which will serve to prevent or decrease expression of the native gene. This position will depend on various factors such as the position of the restriction sites within the sequence to be cut, and whether an exon sequence or a promoter sequence, or both is (are) to be interrupted (i.e., the precise location of insertion necessary to inhibit promoter function or to inhibit synthesis of the native exon) .
  • the enzyme selected for cutting the DNA will generate a longer arm and a shorter arm, where the shorter arm is at least about 300 base pairs (bp) in length.
  • the shorter arm is at least about 300 base pairs (bp) in length.
  • the genomic DNA is cut with appropriate restriction endonucleases such that a fragment of the proper size can be removed. The remaining pieces are ligated with the marker sequence, as discussed below.
  • the marker gene can be any nucleotide sequence that is detectable and/or assayable, however typically it is an antibiotic resistance gene or other gene whose expression or presence in the genome can easily be detected.
  • the marker gene is usually operably linked to its own promoter or to another strong promoter from any source that will be active or can easily be activated in the cell into which it is inserted; however, the marker gene need not have its own promoter attached as it may be transcribed under the control of the promoter of the gene to be suppressed.
  • the marker gene will normally have a polyadenylation (polyA) sequence attached to its 3' end; this sequence serves to terminate transcription of the gene.
  • Preferred marker genes are any antibiotic resistance genes such as neo (the neomycin resistance gene) and beta-gal (beta- galactosidase) .
  • the marker gene sequence is ligated into it using methods well known to the skilled artisan, such as those described in Sambrook et al . , supra .
  • the ends of the DNA fragments to be ligated together must be compatible; this is achieved by either cutting the fragments with enzymes that generate compatible ends, or by blunting the ends prior to ligation. Blunting is conducted using any of several methods well known in the art, such as for example by the use of Klenow fragment (DNA polymerase I) to fill in sticky ends.
  • the ligated knockout construct may be inserted directly into embryonic stem cells (discussed below) , or it may first be placed into a suitable vector for amplification prior to insertion.
  • Preferred vectors are those that are rapidly amplified in bacterial cells such as the pBluescript II SK vector (Stratagene, San Diego, CA) or pGEM7 (Promega Corp., Madison, WI) .
  • This invention contemplates production of knockout mammals from any species of non-human mammal including without limitation, rodents such as rats, hamsters, and mice.
  • rodents such as rats, hamsters, and mice.
  • Preferred rodents include members of the Muridae family, including rats and mice.
  • the embryonic stem cells (ES cells) used to produce the knockout mammal will be of the same species as the knockout mammal to be generated.
  • mouse embryonic stem cells will usually be used for generation of knockout mice.
  • Embryonic stem cells are generated and maintained using methods well known to the skilled artisan such as those described by Doetschman et al . (J. Embryol . Exp. Morphol . 87:27-45 [1985]) .
  • Any line of ES cells can be used, however, the line chosen is typically selected for the ability of the cells to integrate into and become part of the germ line of a developing embryo so as to create germ line transmission of the knockout construct.
  • any ES cell line that is believed to have this capability is suitable for use herein.
  • One mouse strain that is typically used for production of ES cells is the 129J strain.
  • a preferred ES cell line is murine cell line D3 (American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852, catalog no. CRL 1934) .
  • the cells are cultured and prepared for knockout construct insertion using methods well known to the skilled artisan such as those set forth by Robertson (in: Teratocarcinomas and Embryonic Stem Cells : A Practical Approach, E.J. Robertson, ed. IRL Press, Washington, D.C. [1987]) by Bradley et al . (Current
  • Insertion of the knockout construct into the ES cells can be accomplished using a variety of methods well known in the art including for example, electroporation, microinjection, and calcium phosphate treatment (see Lovell-Badge, in Robertson, ed., supra) .
  • a preferred method of insertion is electroporation.
  • Each knockout construct to be inserted into the cell must first be in the linear form. Therefore, if the knockout construct has been inserted into a vector, linearization is accomplished by digesting the DNA with a suitable restriction endonuclease selected to cut only within the vector sequence and not within the knockout construct sequence.
  • the knockout construct is added to the ES cells under appropriate conditions for the insertion method chosen, as is known to the skilled artisan. Where more than one construct is to be introduced into the ES cell, each knockout construct can be introduced simultaneously or one at a time.
  • the ES cells and knockout construct DNA are exposed to an electric pulse using an electroporation machine and following the manufacturer's guidelines for use. After electroporation, the ES cells are typically allowed to recover under suitable incubation conditions. The cells are then screened for the presence of the knockout construct.
  • the ES cells may be cultured in the presence of an otherwise lethal concentration of antibiotic. Those ES cells that survive have presumably integrated the knockout construct.
  • the marker gene is other than an antibiotic resistance gene, a Southern blot of the ES cell genomic DNA can be probed with a sequence of DNA designed to hybridize only to the marker sequence. Alternatively, PCR can be used.
  • the marker gene is a gene that encodes an enzyme whose activity can be detected (e.g., beta-galactosidase)
  • the enzyme substrate can be added to the cells under suitable conditions, and the enzymatic activity can be analyzed.
  • the knockout construct may integrate into several locations in the ES cell genome, and may integrate into a different location in each ES cell's genome due to the occurrence of random insertion events.
  • the desired location of insertion is in a complementary position to the DNA sequence to be knocked out. Typically, less than about 1-5 percent of the ES cells that take up the knockout construct will actually integrate the knockout construct in the desired location.
  • total DNA can be extracted from the ES cells using standard methods such as those described by Sambrook et al. , supra .
  • the DNA can then be probed on a Southern blot with a probe or probes designed to hybridize in a specific pattern to genomic DNA digested with (a) particular restriction enzyme(s) .
  • the genomic DNA can be amplified by PCR with probes specifically designed to amplify DNA fragments of a particular size and sequence (i.e., only those cells containing the knockout construct in the proper position will generate DNA fragments of the proper size) .
  • the cells can be inserted into an embryo. Insertion may be accomplished in a variety of ways known to the skilled artisan, however a preferred method is by microinjection. For microinjection, about 10-30 cells are collected into a micropipet and injected into embryos that are at the proper stage of development to permit integration of the foreign ES cell containing the knockout construct into the developing embryo. The suitable stage of development for the embryo used for insertion of ES cells is very species dependent, however for mice it is about 3.5 days. The embryos are obtained by perfusing the uterus of pregnant females. Suitable methods for accomplishing this are known to the skilled artisan, and are set forth by Bradley (in Robertson, ed., supra) .
  • preferred embryos are male.
  • the preferred embryos also have genes coding for a coat color that is different from the coat color encoded by the ES cell genes.
  • the offspring can be screened easily for the presence of the knockout construct by looking for mosaic coat color (indicating that the ES cell was incorporated into the developing embryo) .
  • the embryo selected will carry genes for black or brown fur.
  • the embryo may be implanted into the uterus of a pseudopregnant foster mother for gestation. While any foster mother may be used, the foster mother is typically selected for her ability to breed and reproduce well, and for her ability to care for the young. Such foster mothers are typically prepared by mating with vasectomized males of the same species.
  • the stage of the pseudopregnant foster mother is important for successful implantation, and it is species dependent. For mice, this stage is about 2-3 days pseudopregnant.
  • Offspring that are born to the foster mother may be screened initially for mosaic coat color where the coat color selection strategy (as described above) has been employed.
  • DNA from tail tissue of the offspring may be screened for the presence of the knockout construct using Southern blots and/or PCR as described above.
  • Offspring that appear to be mosaics may then be crossed to each other, if they are believed to carry the knockout construct in their germ line, in order to generate homozygous knockout animals. If it is unclear whether the offspring will have germ line transmission, they can be crossed with a parental or other strain, and the offspring can be screened for heterozygosity. The heterozygotes are identified by Southern blots and/or PCR amplification of the DNA, as set forth above.
  • the heterozygotes can then be crossed with each other to generate homozygous knockout offspring.
  • Homozygotes may be identified by Southern blotting of equivalent amounts of genomic DNA from mice that are the product of this cross, as well as mice that are known heterozygotes and wild type mice. Probes to screen the Southern blots can be designed as set forth above.
  • Northern blots can be used to probe the mRNA for the presence or absence of transcripts encoding either the gene knocked out, the marker gene, or both.
  • Western blots can be used to assess the level of expression of the gene knocked out in various tissues of these offspring by probing the Western blot with an antibody against the protein encoded by the gene knocked out, or an antibody against the marker gene product, where this gene is expressed.
  • in situ analysis such as fixing the cells and labeling with antibody
  • FACS fluorescence activated cell sorting
  • the transgene(s) useful in the present invention will be a nucleotide sequence encoding a polypeptide involved in the immune response, hematopoiesis, inflammation, cell growth and proliferation, cell lineage differentiation, and/or the stress response.
  • Preferred transgenes are those that comprise polypeptides of the human immune system, such as CD4, and any allelic forms of the HLA DQ locus.
  • the most preferred transgenes are human CD4 and the HLA DQ locus allele DQw6 (both the alpha and beta chains) . Included within the scope of this invention is the insertion of two or more transgenes into a mammal.
  • transgenes useful in this invention may be prepared and inserted individually, or may be generated together as one construct for insertion.
  • the transgenes may be homologous or heterologous to both the promoter selected to drive expression of each transgene and/or to the mammal.
  • the transgene may be a full length cDNA or genomic DNA sequence, or any fragment, subunit or mutant thereof that has at least some biological activity i.e., exhibits an effect at any level
  • the transgene may be a hybrid nucleotide sequence, i.e., one constructed from homologous and/or heterologous cDNA and/or genomic DNA fragments.
  • the transgene may also optionally be a mutant of one or more naturally occurring cDNA and/or genomic sequences, or an allelic variant thereof.
  • Each transgene may be isolated and obtained in suitable quantity using one or more methods that are well known in the art. These methods and others useful for isolating a transgene are set forth,- for example, in Sambrook et ai. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY [1989]) and in Berger and Kimmel (Methods in Enzymology: Guide to Molecular Cloning Techniques, vol. 152, Academic Press, Inc., San Diego, CA [1987]) .
  • the transgene may be synthesized, in whole or in part, using chemical synthesis methods such as those described in Engels et al . (Angew. Chem. Int . Ed. Engl . , 28:716-734 [1989]) . These methods include, inter alia, the phosphotriester, phosphoramidite and H- phosphonate methods of nucleic acid synthesis .
  • the transgene may be obtained by screening an appropriate cDNA or genomic library using one or more nucleic acid probes (oligonucleotides, cDNA or genomic DNA fragments with an acceptable level of homology to the transgene to be cloned, and the like) that will hybridize selectively with the transgene DNA.
  • nucleic acid probes oligonucleotides, cDNA or genomic DNA fragments with an acceptable level of homology to the transgene to be cloned, and the like
  • Another suitable method for obtaining a transgene is the polymerase chain reaction (PCR) .
  • PCR polymerase chain reaction
  • successful use of this method requires that enough information about the nucleotide sequence of the transgene be available so as to design suitable oligonucleotide primers useful for amplification of the appropriate nucleotide sequence.
  • the oligonucleotide sequences selected as probes or primers should be of adequate length and sufficiently unambiguous so as to minimize the amount of non-specific binding that will occur during library screening or PCR.
  • the actual sequence of the probes or primers is usually based on conserved or highly homologous sequences or regions from the same or a similar gene from another organism.
  • the probes or primers can be degenerate.
  • a probable and functional nucleic acid sequence may be inferred for the transgene using known and preferred codons for each amino acid residue. This sequence can then be chemically synthesized.
  • a mutant transgene is a transgene containing one or more nucleotide substitutions, deletions, and/or insertions as compared to the wild type sequence.
  • the nucleotide substitution, deletion, and/or insertion can give rise to a gene product (i.e., protein) that is different in its amino acid sequence from the wild type amino acid sequence.
  • Preparation of such mutants is well known in the art, and is described for example in Wells et al . (Gene, 34:315 [1985]), and in Sambrook et al, supra .
  • transgenes of the present invention are typically operably linked to promoters, where a promoter is selected to regulate expression of each transgene in a particular manner.
  • Each transgene may be regulated by the same or by a different promoter.
  • the selected promoters may be homologous (i.e., from the same species as the mammal to be transfected with the transgene) or heterologous (i.e., from a source other than the species of the mammal to be transfected with the transgene) .
  • the source of each promoter may be from any unicellular, prokaryotic or eukaryotic organism, or any vertebrate or invertebrate organism.
  • the more preferred promoters of this invention are human and mouse promoters that regulate expression of genes of the immune system such as the human CD2 promoter, the human CD4 promoter, the HLA DQw6 alpha and beta promoters, the mouse CD4 promoter, the mouse p56 lck promoter, the mouse I-E alpha promoter, and the mouse H-2 ⁇ promoter.
  • the most preferred promoters are the human CD2 promoter and the HLA DQw6 alpha and beta promoters .
  • the promoters of this invention may be used alone or in combination with homologous and/or heterologous enhancers and/or silencers in order to permit a tighter regulation of expression.
  • the nucleotide sequences of the promoters of this invention may be obtained by any of several methods well known in the art. Typically, promoters useful herein will have been previously identified by mapping and/or by restriction endonuclease digestion and can thus be isolated from the genomic DNA of the proper tissue source using the appropriate restriction endonucleases. In some cases, the promoter may have been sequenced. For those promoters whose nucleotide sequence is known, the promoter may be synthesized using the methods described above for transgene synthesis. Where all or only portions of the promoter sequence are known, the promoter may be obtained using PCR and/or by screening a genomic library with suitable oligonucleotide and/or promoter sequence fragments from the same or another species.
  • a fragment of DNA containing the promoter may be isolated from a larger piece of DNA that may contain, for example, a coding sequence or even another gene or genes. Isolation may be accomplished by restriction endonuclease digestion using one or more carefully selected enzymes to isolate the proper DNA fragment. After digestion, the desired fragment is isolated by agarose gel purification, Qiagen® column (Qiagen Corp., Chatsworth, CA) or other methods known to the skilled artisan. Selection of suitable enzymes to accomplish this purpose will be readily apparent to one of ordinary skill in the art.
  • the vectors useful for preparing the transgenes of this invention typically contain one or more other elements useful for (1) optimal expression of transgene in the mammal into which the transgene is inserted, and (2) amplification of the vector in bacterial or mammalian host cells.
  • Each of these elements will be positioned appropriately in the vector with respect to each other element so as to maximize their respective activities. Such positioning is well known to the ordinary skilled artisan.
  • the following elements may be optionally included in the vector as appropriate.
  • a small polypeptide termed signal sequence is frequently present to direct the polypeptide encoded by the transgene out of the cell where it is synthesized.
  • the signal sequence is positioned in the coding region of the transgene towards or at the 5 ' end of the coding region.
  • Many signal sequences have been identified, and any of them that are functional and thus compatible with the transgenic tissue may be used in conjunction with the transgene. Therefore, the nucleotide sequence encoding the signal sequence may be homologous or heterologous to the transgene, and may be homologous or heterologous to the transgenic mammal.
  • nucleotide sequence encoding the signal sequence may be chemically synthesized using methods set forth above. However, for purposes herein, preferred signal sequences are those that occur naturally with the transgene (i . e . , are homologous to the transgene) .
  • transgene expressed on the surface of a particular intracellular membrane or on the plasma membrane it may be desirable to have a transgene expressed on the surface of a particular intracellular membrane or on the plasma membrane.
  • Naturally occurring membrane proteins contain, as part of the polypeptide, a stretch of amino acids that serve to anchor the protein to the membrane.
  • the anchor domain will be an internal portion of the polypeptide sequence and thus the nucleotide sequence encoding it will be engineered into an internal region of the transgene nucleotide sequence.
  • the nucleotide sequence encoding the anchor domain may be attached to the 5' or 3' end of the transgene nucleotide sequence.
  • the nucleotide sequence encoding the anchor domain may first be placed into the vector in the appropriate position as a separate component from the nucleotide sequence encoding the transgene.
  • the anchor domain may be from any source and thus may be homologous or heterologous with respect to both the transgene and the transgenic mammal.
  • the anchor domain may be chemically synthesized using methods set forth above. iii. Origin of Replication Element
  • This component is typically a part of prokaryotic expression vectors purchased commercially, and aids in the amplification of the vector in a host cell. If the vector of choice does not contain an origin of replication site, one may be chemically synthesized based on a known sequence, and ligated into the vector.
  • This element also known as the polyadenylation or polyA sequence, is typically located 3' to the transgene nucleotide sequence in the vector, and serves to terminate transcription of the transgene. While the nucleotide sequence encoding this element is easily cloned from a library or even purchased commercially as part of a vector, it can also be readily synthesized using methods for nucleotide sequence synthesis such as those described above.
  • transcription of the transgene is increased by the presence of one or more introns on the vector.
  • the intron may be naturally occurring within the transgene nucleotide sequence, especially where the transgene is a full length or a fragment of a genomic DNA sequence. Where the intron is not naturally occurring within the nucleotide sequence (as for most cDNAs) , the intron(s) may be obtained from another source.
  • the intron may be homologous or heterologous to the transgene and/or to the transgenic mammal. The position of the intron with respect to the promoter and the transgene is important, as the intron must be transcribed to be effective.
  • the preferred position for the intron is 3' to the transcription start site, and 5' to the polyA transcription termination sequence.
  • the intron will be located on one side or the other (i . e . , 5' or 3') of the transgene nucleotide sequence such that it does not interrupt the transgene nucleotide sequence.
  • Any intron from any source including any viral, prokaryotic and eukaryotic (plant or animal) organisms, may be used to practice this invention, provided that it is compatible with the host cell(s) into which it is inserted.
  • synthetic introns may be used in the vector.
  • Selectable marker genes encode proteins necessary for the survival and growth of transfected cells grown in a selective culture medium.
  • Typical selection marker genes encode proteins that (a) confer resistance to antibiotics or other toxins, e . g. , ampicillin, tetracycline, or kanomycin for prokaryotic host cells, and neomycin, hygromycin, or methotrexate for mammalian cells; (b) complement auxotrophic deficiencies of the cell; or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for cultures of Bacilli .
  • the vectors most useful for preparation of transgenic mammals of this invention are those that are compatible with prokaryotic cell hosts.
  • eukaryotic cell hosts, and vectors compatible with these cells are within the scope of the invention.
  • some of the various vector elements may be already present in commercially available vectors such as pUC18, pUC19, pBR322, the pGEM vectors (Promega Corp, Madison, WI) , the pBluescript® vectors such as pBIISK+/- (Stratagene Corp., La Jolla, CA) , and the like, all of which are suitable for prokaryotic cell hosts. In this case it is necessary to only insert the transgene(s) into the vector.
  • the elements to be used are not already present in the vector, they may be individually obtained and ligated into the vector. Methods used for obtaining each of the elements and ligating them are well known to the skilled artisan and are comparable to the methods set forth above for obtaining a transgene (i.e., synthesis of the DNA, library screening, and the like) .
  • Vectors used for amplification of the transgene (s) nucleotide sequences and/or for transfection of the mammalian embryos are constructed using methods well known in the art. Such methods include, for example, the standard techniques of restriction endonuclease digestion, ligation, agarose and acrylamide gel purification of DNA and/or RNA, column chromatography purification of DNA and/or RNA, phenol/chloroform extraction of DNA, DNA sequencing, polymerase chain reaction amplification, and the like, as set forth in Sambrook et al . , supra .
  • the final vector used to practice this invention is typically constructed from a starting vector such as a commercially available vector.
  • This vector may or may not contain some of the elements to be included in the completed vector. If none of the desired elements are present in the starting vector, each element may be individually ligated into the vector by cutting the vector with the appropriate restriction endonuclease (s) such that the ends of the element to be ligated in and the ends of the vector are compatible for ligation. In some cases, it may be necessary to "blunt" the ends to be ligated together in order to obtain a satisfactory ligation. Blunting is accomplished by first filling in "sticky ends" using Klenow DNA polymerase or T4 DNA polymerase in the presence of all four nucleotides. This procedure is well known in the art and is described for example in Sambrook et al . , supra .
  • two or more of the elements to be inserted into the vector may first be ligated together (if they are to be positioned adjacent to each other) and then ligated into the vector.
  • One other method for constructing the vector is to conduct all ligations of the various elements simultaneously in one reaction mixture.
  • many nonsense or nonfunctional vectors will be generated due to improper ligation or insertion of the elements, however the functional vector may be identified and selected by restriction endonuclease digestion.
  • the vector After the vector has been constructed, it may be transfected into a prokaryotic host cell for amplification.
  • Cells typically used for amplification are E coli DH5-alpha (Gibco/BRL, Grand Island, NY) and other E. coli strains with characteristics similar to DH5-alpha.
  • cell lines such as Chinese hamster ovary (CHO cells; Urlab et al . , Proc . Natl . Acad. Sci USA, 77:4216 [1980])) and human embryonic kidney cell line 293 (Graham et al . , J. Gen . Virol . , 36:59 [1977]), as well as other lines, are suitable.
  • Transfection of the vector into the selected host cell line for amplification is accomplished using such methods as calcium phosphate, electroporation, microinjection, lipofection or DEAE-dextran.
  • the method selected will in part be a function of the type of host cell to be transfected.
  • the vector (often termed plasmid at this stage) is isolated from the cells and purified. Typically, the cells are lysed and the plasmid is extracted from other cell contents. Methods suitable for plasmid purification include inter alia, the alkaline lysis mini-prep method (Sambrook et al . , supra) .
  • the plasmid containing the transgene is linearized using a selected restriction endonuclease prior to insertion into the embryo.
  • the specific line(s) of any mammalian species used to practice this invention are selected for general good health, good embryo yields, good pronuclear visibility in the embryos, and good reproductive fitness.
  • the haplotype is a significant factor. For example, when transgenic mice are to be produced, strains such as C57BL/6 or C57BL/6 x DBA/2 Fi, or FVB lines are often used (obtained commercially from Charles River Labs, Boston, MA, The Jackson Laboratory, Bar Harbor, ME, or Taconic Labs.) . Preferred strains are those with H-2 b ' H-2 d or H-2 0 . haplotypes such as C57BL/6 or DBA/1.
  • the line(s) used to practice this invention may themselves be transgenics, and/or may be knockouts (i.e., mammals which have one or more genes partially or completely suppressed) .
  • the same line will be used for preparation of both the initial knockout mammals and the transgenic mammals. This will make subsequent breeding and backcrossing more efficient.
  • the age of the mammals that are used to obtain embryos and to serve as surrogate hosts is a function of the species used, but is readily determined by one of ordinary skill in the art. For example, when mice are used, pre-puberal females are preferred, as they yield more embryos and respond better to hormone injections. Similarly, the male mammal to be used as a stud will normally be selected by age of sexual maturity, among other criteria.
  • hormones or other chemical compounds may be necessary to prepare the female for egg production, mating, and/or reimplantation of embryos.
  • the type of hormones/cofactors and the quantity used, as well as the timing of administration of the hormones will vary for each species of mammal. Such considerations will be readily apparent to one of ordinary skill in the art
  • a primed female i.e., one that is producing eggs that can be fertilized
  • a stud male i.e., one that is producing eggs that can be fertilized
  • the resulting fertilized embryos are then removed for introduction of the transgene(s) .
  • eggs and sperm may be obtained from suitable females and males and used for in vitro fertilization to produce an embryo suitable for introduction of the transgene. Normally, fertilized embryos are incubated in suitable media until the pronuclei appear. At about this time, the nucleotide sequence comprising the transgene is introduced into the female or male pronucleus as described below. In some species such as mice, the male pronucleus is preferred.
  • transgene nucleotide sequence into the embryo may be accomplished by any means known in the art such as, for example, microinjection, electroporation, or lipofection.
  • the embryo may be incubated in vitro for varying amounts of time, or reimplanted into the surrogate host, or both. In vitro incubation to maturity is within the scope of this invention.
  • One common method is to incubate the embryos in vitro for about 1-7 days, depending on the species, and then reimplant them into the surrogate host.
  • Reimplantation is accomplished using standard methods. Usually, the surrogate host is anesthetized, and the embryos are inserted into the oviduct. The number of embryos implanted into a particular host will vary by species, but will usually be comparable to the number of offspring the species naturally produces.
  • Transgenic offspring of the surrogate host may be screened for the presence and/or expression of the transgene by any suitable method. Screening is often accomplished by Southern blot or Northern blot analysis, using a probe that is complementary to at least a portion of the transgene. Western blot analysis using an antibody against the protein encoded by the transgene may be employed as an alternative or additional method for screening for the presence of the transgene product. Typically, DNA is prepared from tail tissue (about 1 cm is removed from the tip of the tail) and analyzed by Southern analysis or PCR for the transgene.
  • the tissues or cells believed to express the transgene at the highest levels are tested for the presence and expression of the transgene using Southern analysis or PCR, although any tissues or cell types may be used for this analysis.
  • transgenic mammals may be obtained by mating the transgenic mammal with a suitable partner, or by in vitro fertilization of eggs and/or sperm obtained from the transgenic mammal.
  • the partner may or may not be transgenic and/or a knockout; where it is transgenic, it may contain the same or a different transgene, or both.
  • the partner may be a parental line.
  • in vitro fertilization the fertilized embryo may be implanted into a surrogate host or incubated in vitro, or both. Using either method, the progeny may be evaluated for the presence of the transgene using methods described above, or other appropriate methods.
  • Mammals containing more than one knockout construct and/or more than one transgene are prepared in any of several ways.
  • the preferred manner of preparation is to generate a series of mammals, each containing one of the desired knockout constructs or transgenes. Such mammals are bred together through a series of crosses, backcrosses and selections, to ultimately generate a single mammal containing all desired knockout constructs and/or transgenes, where the mammal is otherwise congenic (genetically identical) to the wild type except for the presence of the knockout (s) constructs and/or transgene(s) .
  • Figure 4 depicts a breeding scheme for generating a mouse that is a CD4,
  • CD8 double knockout (mCD4-/-, mCD8-/-) and contains two transgenes (human DQw6 alpha and beta chains [DQw6], and human CD4 [hCD4] ) .
  • crossing and backcrossing is accomplished by mating siblings or a parental strain with an offspring, depending on the goal of each particular step in the breeding process.
  • the murine genes encoding CD4 and CD8 are located relatively close together on the same chromosome.
  • to generate a mouse that has both CD4 and CD8 knocked out one has essentially two practical choices.
  • the mammal and its progeny of this invention will have a variety of uses depending on the transgenes expressed and the knockout constructs they contain.
  • the mammal may be used to screen for drugs or a therapeutic regimen useful for prophylactic or therapeutic treatment of diseases such as sepsis or other immunological disorders. Screening for a useful drug would involve first inducing the disease in the mammal (i . e . , exposing the mammal to a pathogen or toxin causing sepsis) and then administering the candidate drug over a range of doses to the mammal, and assaying at various time points for the effect (s) of the drug on the disease or disorder being evaluated. Alternatively, or additionally, the drug could be administered prior to or simultaneously with exposure to induction of the disease.
  • the mammal of the present invention could be useful in designing a therapeutic regimen aimed at preventing or curing the disease or condition.
  • the mammal might be treated with a combination of a particular diet, exercise routine, radiation treatment, and/or one or more compounds or substances either prior to, or simultaneously, or after, the onset of the disease or condition.
  • Such an overall therapy or regimen might be more effective at combating the disease or condition than treatment with a compound alone.
  • Assays to evaluate the efficacy of the compound and/or therapeutic regimen would include, for example, looking for increased or decreased T and B cell levels, increased or decreased immunoglobulin production, increased or decreased levels of chemical messengers such as cytokines (e . g.
  • tumor necrosis factor and the like and/or increased or decreased levels of expression of particular genes involved in the immune response.
  • such criteria as blood pressure, body temperature, body weight, pulse, behavior, appearance of coat (ruffled fur) and the like could be evaluated.
  • a mammal of the present invention could be used to screen a variety of compounds, either alone or in combination, to determine whether such reduction in disease symptoms could be decreased or alleviated.
  • mammals of the present invention can be useful for evaluating the development of the immune system, and for studying the effects of particular gene mutations.
  • the transgenic knockout mammals of this invention may also be used to generate one or more cell lines.
  • Such cell lines have many uses, as for example, to evaluate the effect (s) of the transgene knockout on a particular tissue or organ, and to screen compounds that may affect the level of activity of the transgene in the tissue. Such compounds may be useful as therapeutics to modulate the activity of the transgene.
  • Production of such cell lines may be accomplished using a variety of methods, known to the skilled artisan.
  • the actual culturing conditions will depend on the tissue and type of cells to be cultured.
  • Various media containing different concentrations of macro and micro nutrients, growth factors, serum, and the like, can be tested on the cells without undue experimentation to determine the optimal conditions for growth and proliferation of the cells.
  • other culturing conditions such as cell density, media temperature, and carbon dioxide concentrations in the incubator can also readily be evaluated, repair, and identifying compounds that affect this process.
  • Other uses of the invention will be readily apparent to one skilled in the art.
  • 2/neo construct was linearized by restriction endonuclease digestion, and then electroporated into D3 murine embryonic stem cells (described by Doetschman et al . , J. E bryol . Exp . Morph . , 87:27-45 [1985]) using the procedure of electroporation.
  • electroporation about 5 nmol of the linearized construct in was added to about 5 X 106 ES cells in a volume of about 800 ⁇ l of culture medium. The cells were pulsed at about 0.34 kilovolts and 250 ⁇ F, and each vial of cells was then placed on two 10 cm cell culture plates containing embryonic fibroblast feeder cells .
  • the culture plates were pre-coated with 1 percent gelatin, and contained 10 ml DMEM medium (Gibco/BRL, Grand Island, NY) , with 15 percent fetal calf serum (Gibco/BRL, Grand Island, NY, or the equivalent from Hyclone Labs, Logan, UT) , and leukemia inhibitory factor (Fung-Leung et al . , Cell,
  • neo selection was started by adding the antibiotic G418 at about 250-300 ⁇ g/ml to the cultures, and the media was changed approximately every two days.
  • Cells that survived in the presence of G418 most likely contained the knockout construct with the neo gene in the proper orientation for expression. These cells were then screened to verify that they had incorporated the knockout construct into their DNA. Screening was accomplished using PCR.
  • the D3 cells containing the Lyt-2/neo knockout construct have been deposited with the American Type Culture Collection as accession number CRL-11116.
  • the embryos injected were 3.5 day old embryos obtained by perfusing the uterus of female mice that had been mated with male mice. After injection of the embryonic stem cells into the embryos, the embryos were implanted into pseudopregnant females for the duration of gestation. The offspring were mated to either each other or to a mouse with suitable coat color so as to be able to detect mice carrying the knockout construct.
  • mice were evaluated for the presence of the knockout construct by probing a Southern blot of DNA obtained from tail tissue with probes designed to detect the neo gene.
  • the neo-poly A insert was isolated and was then ligated into the CD4 construct previously digested with Kpnl, which cuts in an internal site of exon 5 in the antisense orientation relative to the CD4 transcriptional orientation.
  • the resulting knockout construct is depicted in Figure 2.
  • the construct was amplified in E coli cells and the plasmid containing the construct was then purified.
  • the knockout construct was linearized by restriction endonuclease digestion, and the DNA was then purified. About 5 nmol of this DNA was then electroporated into D3 embryonic stem cells and the electroporated cells selected and screened as described in Example 1.
  • the D3 cell line containing the CD4 knockout construct has been deposited with The American Type Culture Collection as Accession Number CRL-11114.
  • the cells were microinjected into 3.5 day old murine embryos; the embryos were obtained from females that had been mated with males. The embryos were then implanted into pseudopregnant foster mothers for the duration of gestation. The offspring of these mice were evaluated for the presence of the knockout construct by probing a Southern blot of DNA obtained from tail tissue.
  • the CD4 and CD8 knockout mice described above were used to generate a mCD4-/-/mCD8-/- double knockout mouse.
  • mCD4+/-mCD8+/+ mice were bred with mCD4+/+mCD8- /- mice to obtain mCD4+/-mCD8+/- heterozygous Fi offspring.
  • the heterozygote siblings were crossed, and the offspring were screened as follows.
  • About 20 ⁇ l of blood was obtained from the tail vein of each mouse and was collected into heparinized capillary tubes. The blood was washed once in Immunofluoescence Staining Buffer ("IBS", consisting of calcium and magnesium free phosphate buffered saline [Sambrook et al .
  • IBS Immunofluoescence Staining Buffer
  • the cells were then analyzed by flow cytometric analysis using Lysis II software (Becton- Dickinson Co.) . Wild-type and heterozygote CD4/CD8 knockout mutants were distinguished from each other on the basis of intensity of staining; homozygous CD4/CD8 knockout mutants were identified by the complete absence of either CD4+ or CD8+ cells (based on the lack of immunofluroescence staining with the antibodies) . The frequency of offspring homozygous for the mutation on both loci was 2 percent (4 out of 200) .
  • mice The human CD4 (hCD4) transgenic mice were prepared as follows:
  • the final construct as depicted in Figure 3 is copy number dependent and is specifically expressed in lymphocytes, regardless of the integration site in the genome of the transgenic mouse.
  • This construct was injected into CBA/Ca x C57BL/10 embryos using the procedures described above.
  • the offspring were screened for the presence of the transgene construct, and those carrying the construct were backcrossed to C57BL/6 mice for five generations.
  • the offspring of the F5 generation were then bred into the CD4/CD8 double knockout mice to generate mice that are CD4/CD8 double knockouts containing the human CD4 transgene.
  • a C57B46 mouse carrying the transgenes human HLA-DQw6 alpha and HLA-DQw6 beta was prepared as follows.
  • the gene encoding the HLA-DQw6 alpha chain was isolated from a genomic library of DNA prepared from a human B lymphoblastoid cell line (EB-TOK) previously transformed with EBV (Epstein-Barr virus) , using lambda charon 4A as a cloning vector. This cell line is homozygous for the HLA-DR2-DQw6-Dwl2 haplotype.
  • the gene was obtained as an approximately 13 kb EcoRI fragment containing about 0.8kb of 5' sequence, the full length coding region, and about 6kb of 3' untranslated sequence.
  • the gene encoding the DQw6 beta chain was isolated as described by Tsukamoto et al . ( Immunogenetics, 25:343-346 [1987]) . An approximately 18kb EcoRI fragment of the DQw6 beta chain nucleotide sequence was ligated to a EcoRI-Pstl cDNA sequence containing exon 6 of DQw6 beta and some 3' untranslated sequence in order to generate a complete DQw6 beta gene.
  • DQw6 alpha and beta genes were linearized by restriction endonuclease digestion with EcoRI.
  • a mixture of the DQw6 alpha and beta genes was then microinjected into embryos of C57B46 mice.
  • the embryos were implanted into a foster mother for the duration of gestation.
  • the pups were screened for the presence of the transgenes as follows.
  • Tail tissue was obtained from each mouse and DNA from the tissue was prepared using the SDS-proteinase K method.
  • the DNA was digested with BamHI and then subjected to electrophoresis on a 0.9% agarose gel. Southern blot analysis was then performed to identify those pups containing the transgene.
  • MHCII transgenic mice were then backcrossed to C57BL/6 mice for ten generations.
  • the offspring of the F10 generation were then crossed with each other to obtain homozygous DQw6+/+ transgenic mice.
  • the homozygotes were then bred with the CD4/CD8 double knockout mice to obtain mice that are CD4/CD8 double knockouts and contain the DQw6 alpha and beta transgenes .
  • the CD4/CD8 double knockout mice containing the human CD4 transgene were bred with the CD4/CD8 double knockout mice containing the DQw6 transgenes to obtain offspring that are double knockout, double transgenic mice.
  • the mice were typed for the presence of the knockout constructs and transgenes using the immunofluorescence staining procedures set forth above. The complete breeding scheme used to generate this jouse is depicted in Figure 5.
  • Example 7 Screening for Sepsis Susceptibility
  • mice The effects of SEB (Staphylococcus enterotoxin B, Sigma Chemical Co., St. Louis, MO), LPS (lipopolysaccharide, Sigma Chemical Co.), and anti CD3 monoclonal antibody (Pharmingen, San Diego, CA) were evaluated in wild type, transgenic, knockout, and double knockout, double transgenic mice, all of which were between 6 and 12 weeks old. Specifically, the following mice were evaluated:
  • mouse CD4-/-, mouse CD8-/- (mCD4-/-,mCD8-/-)
  • Wild-type rodents are generally resistant to the effects of enterotoxins. Therefore, administration of about 20mg (in 100 ⁇ l PBS) of the sensitizing agent D-galactosamine (D-gal) was given to each mouse intraperitoneally about 5-15 minutes prior to injection of the test substance. Each test substance was administered intraperitoneally in about 100 ⁇ l of PBS. The amount of each test substance that was administered is indicated in Table 1.
  • mice The number of mice that died within 72 hours after exposure to SEB or LPS is shown in Table 1.
  • the double knockout, double transgenic mice showed a very high sensitivity to SEB, as the lethality was very high in these mice as compared with all other genotypes evaluated.
  • T cells of the double knockout double transgenic mice were more responsive to the presence of SEB as compared with the wild type and other mutant genotypes .

Abstract

Mammifère non humain doublement transgénique, et à double expulsion de gène endogène, qui sert de modèle pour évaluer des traitements thérapeutiques ou préventifs de maladies infectieuses.
PCT/IB1994/000417 1993-11-29 1994-11-28 Modele pour maladies infectieuses WO1995014377A2 (fr)

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AU10758/95A AU1075895A (en) 1993-11-29 1994-11-28 Sepsis model
EP95901576A EP0734201A1 (fr) 1993-11-29 1994-11-28 Modele pour maladies infectieuses
JP7514946A JPH09500025A (ja) 1993-11-29 1994-11-28 敗血症モデル

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US15867793A 1993-11-29 1993-11-29
US08/158,677 1993-11-29

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1414949A1 (fr) * 2001-07-13 2004-05-06 Genoway Cellule et animal transgenique modelisant la presentation antigenique humaine et leurs utilisations

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CELL, vol. 65, 3 May 1991 CAMBRIDGE, pages 443-449, MARCO W. SCHILHAM ET AL. 'CD8 is needed for development of cytotoxic T cells but not helper T cells' cited in the application *
EMBO JOURNAL, vol. 12, no. 4, 1993 OXFORD, pages 1547-1553, NIGEL KILLEEN 'Regulated expression of human CD4 rescues helper T cell development in mice lacking expression of endogenous CD4' cited in the application *
EUROP. J. IMMUNOLOGY , vol. 23, June 1993 pages 1299-1304, MARCO W. SCHILHAM ET AL. 'Alloreactive cytotoxic T cells can develop and fumction in mice lacking both CD4 and CD8' cited in the application *
MOLECULAR CELLULAR BIOL., vol. 13, no. 5, May 1993 pages 2952-2958, F. P. GILLESPIE ET AL. 'Tissue-specific expression of human CD4 in transgenic mice' *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1414949A1 (fr) * 2001-07-13 2004-05-06 Genoway Cellule et animal transgenique modelisant la presentation antigenique humaine et leurs utilisations

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EP0734201A1 (fr) 1996-10-02
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WO1995014377A3 (fr) 1995-08-31
AU1075895A (en) 1995-06-13

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