EP0789757A1 - Organismes transgeniques et leurs utilisations - Google Patents

Organismes transgeniques et leurs utilisations

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Publication number
EP0789757A1
EP0789757A1 EP95936026A EP95936026A EP0789757A1 EP 0789757 A1 EP0789757 A1 EP 0789757A1 EP 95936026 A EP95936026 A EP 95936026A EP 95936026 A EP95936026 A EP 95936026A EP 0789757 A1 EP0789757 A1 EP 0789757A1
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Prior art keywords
cells
tissue
cell
selectable marker
transgenic
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EP95936026A
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German (de)
English (en)
Inventor
Bradley Michael John Stringer
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CellFactors PLC
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CellFactors PLC
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Publication of EP0789757A1 publication Critical patent/EP0789757A1/fr
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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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • 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
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • 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
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • 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
    • C12N2517/00—Cells related to new breeds of animals
    • C12N2517/02—Cells from transgenic animals

Definitions

  • the present invention relates to transgenic organisms having cells with one or more selectable phenotypes and their uses.
  • Transgenic organisms including transgenic animals, have been known for a number of years. Although the term has occasionally been applied to any organism which contains foreign DNA, the term "transgenic organism” is used herein in its more usual sense to denote eukaryotic organisms (and in particular, animals or plants, and especially vertebrates e.g. mammals) and their progeny which contain heterologous chromosomal DNA in the germ line.
  • the heterologous chromosomal DNA comprises a coding sequence which is hereinafter referred to as a "transgene".
  • every (or at least most) of the cells of a transgenic organism - both somatic and germ - may contain one or more copies of the transgene(s) .
  • Transgenic organisms can be produced by many different methods. The methods are well documented in the prior art and their practice forms part of the technical repertoire of those skilled in the art. Methodological approaches commonly used are described for example in First and Haseltine (Eds.), Transgenic Animals (1991) Butterworth- Heineman MA USA.
  • the transgene is inserted into embryonic stem cells which are then injected into fertilized zygotes at a stage when only a small number of cells are present.
  • the engineered embryonic stem cells become incorporated into the zygote, and cells derived therefrom go on to differentiate into many or all of the different cell types of the animal's body.
  • Such cells may also include those contributing to the germline, and the progeny of such (chimaeric) animals may- therefore be fully transgenic.
  • selectable markers genes which confer any of a wide variety of selectable phenotypes thereon.
  • selectable markers genes which are known as selectable markers. They are normally introduced into cells as part of a reco binant expression vector. The selectable phenotype conferred by a selectable marker may be classed as either positive or negative.
  • a positive selectable phenotype is one which permits survival under particular conditions which would kill (or at least prevent or impair the growth of) cells which do not exhibit the positive selectable phenotype.
  • a negative selectable phenotype is one which results in the destruction (or the prevention or impairment of growth) of the cell under particular conditions which are relatively innocuous to cells which do not exhibit the negative selectable phenotype.
  • Genes that are widely applied as positive selectable markers include the bacterial neomycin phosphotransferase (neo; Colbere-Garapin et al. (1981) 150:1), hygromycin phosphotransferase (hph; Santerre et al. (1984), Gene 30:147) and xanthineguanine phosphoribosyl transferase (gpt; Mulligan and Berg (1981), P.N.A.S., USA 78:2072).
  • HSV-1 TK Herpes simplex virus type 1 thy idine kinase
  • APRT adenine phosphoribosyl- transferase
  • HPRT hypoxanthine phosphoribosyltransferase
  • Some of the aforementioned genes also confer negative as well as positive selectable phenotypes. They include the HSV-1 TK, APRT, HPRT and gpt genes. These genes encode enzymes which can catalyse the conversion of certain nucleoside or purine analogues to cytotoxic intermediates.
  • the nucleoside analog ganciclovir (GCV) is a good substrate for the HSV-1 thymidine kinase but a poor substrate for the natural thymidine kinase found in mammalian cells. Consequently, GCV can be used for efficient negative selection against cells expressing the HSV-1 TK gene (St. Clair et al. (1987), Antimicrob. Agents Chemother.
  • Xanthineguanine phosphoribosyl transferase can be used for both positive and negative selection when expressed in wild type cells (Besnard et al. (1987), Mol. Cell. Biol. 7:4139).
  • Selectable markers are usually used in both prokaryotic and eukaryotic genetic engineering to permit the recovery, from a mixed population, of cells which have undergone a relatively rare genetic change.
  • they can be used in physical association with another gene which encodes a product of interest (for example, a therapeutic protein) to select cells which have taken up that other gene along with the selectable marker.
  • a product of interest for example, a therapeutic protein
  • the neo gene has been used to monitor genetically modified cells taken from patient samples after gene therapy has taken place.
  • negative selectable markers have been proposed to use negative selectable markers as a safety device in gene therapy.
  • Many gene therapies involve the removal of somatic cells from the patient, the introduction therein of a therapeutic gene (the expression of which repairs a biochemical lesion), followed by reintroduction of the cells back into the patient. Since the reintroduced genetically modified cells may ultimately prove deleterious to the health of the patient (for example, if they prove to be immunologically incompatible or become malignant), a negative selectable marker may be introduced along with the therapeutic gene to permit (if necessary) subsequent selective elimination of the genetically modified cells.
  • a number of vectors bearing positive or negative selectable markers have been made and are readily available to those skilled in the art (for review see Miller (1992), Nature 357:455-460). Others may be readily assembled using standard gene cloning techniques.
  • Transgenic organisms bearing a selectable marker as a transgene are known in the art.
  • the selectable marker is introduced to permit the recovery of cells which have also taken up a gene of interest to which the selectable marker was linked.
  • many such transgenic organisms have been constructed in the course of work involving the introduction into the germline of genetic information which disrupts normal development (see WO/91/13150) .
  • Transgenic organisms bearing a selectable marker have also been constructed in the course of the construction of animals bearing a specific genetic lesion.
  • the selectable marker is inserted (usually by homologous recombination) into a particular gene which is thereby insertionally inactivated.
  • the selectable phenotype conferred by the selectable marker is then used as the basis for the identification and recovery of cells bearing an insertionally inactivated copy of the gene. Examples of transgenic animals produced by such methods are described in Piedrahita et al. (1992), PNAS 89, pages 4471-4475.
  • tissue/cell culture methods are fundamental to pharmaceutical, clinical, agricultural and industrial research.
  • tissue/cell culture methods are used in the pharmaceutical industry for the preparation of medicaments (for example, therapeutically active protein products) and in assays or screens for potential drugs.
  • transgenic organisms bearing positive and/or negative selectable markers have previously unrecognized utility in cell culture techniques.
  • the present invention provides transgenic organisms which inter alia constitute a very convenient source of material for the isolation, identification, culture and analysis of cells from any tissue of the organism's body.
  • Tissue dissected from the transgenic organisms of the invention can be particularly easily grown (even as homogenous populations of a particular cell/tissue type) in vitro and used in a wide variety of applications, including pharmaceutical assays, tissue transplant synthesis, drug delivery and protein production.
  • a transgenic eukaryotic organism having cells containing heterologous DNA comprising a transgene encoding a positive selectable marker and a transgene encoding a negative selectable marker.
  • the organism may be essentially normal, the transgenes for example not being located such that they insertionally inactivate a gene.
  • the term "essentially normal” as used herein may indicate that the organism is not mutant for any significant character or trait with respect to the wild type and/or exhibits normal tissue differentiation and development.
  • the organism may be essentially normal in the sense that the transgenes are resident in a silent (i.e. non-expressed) region of the genome and/or in a region of the genome where the transgenes do not significantly perturb the replication, segregation, organisation or packing of the chromosome or its interaction with cellular components such as DNA binding proteins (including histones and regulatory elements).
  • the provision of transgenes encoding both positive and negative selectable markers provides great flexibility during subsequent manipulation of cells derived from the transgenic organism in vitro.
  • cells of a particular type may be isolated from the transgenic animal of the invention by positive selection.
  • the cells so isolated may then be transplanted into a non- transgenic animal to determine whether the transplant has any therapeutic effect.
  • the transplant may then be ablated by negative selection to provide a control to determine whether the transplant was having a direct therapeutic effect.
  • the invention provides a transgenic eukaryotic organism having cells containing heterologous DNA comprising a transgene encoding a positive selectable marker and/or a transgene encoding a negative selectable marker, the organism being essentially normal but for the selectable phenotypes arising from the transgene(s) .
  • a positive and negative selectable marker may be provided by a single transgene, since (as explained above) some markers can be used as both positive and negative selectable markers (depending upon the selection conditions used) .
  • the transgenic eukaryotic organism of the invention is preferably an animal or a plant, for example a vertebrate (e.g. a mammal, for example a rat, rabbit, pig or mouse).
  • the transgenic organism preferably has a genotype which is essentially wild type but for the presence of the heterologous DNA.
  • that portion of the heterologous DNA which is expressed in the cells may consist of a transgene encoding a positive selectable marker and/or a transgene encoding a negative selectable marker, each transgene being operably linked to an expression element or elements.
  • At least one of the selectable markers may be operably linked to a regulatable expression element or elements, for example a tissue- or cell-specific expression element or elements.
  • each selectable marker is advantageously differentially regulated, each marker for example being linked to a different tissue- or cell-specific expression element or elements. This permits the expression of the selectable marker to be limited to a selected class of cells or tissue, so providing e.g. for the selective culture in vitro of the selected class of cells or tissue from a mixed primary cell culture.
  • the present invention does not rely on the use of transgenic organisms produced by any one method: any transgenic procedure may be used in the practice of the invention. Moreover, in most circumstances, the precise nature of the selectable markers for use in the present invention is unimportant: in general, any selectable marker gene may be used so long as it confers a positive or negative selectable phenotype on the cell.
  • the positive selectable marker may be selected from neomycin phosphotransferase, hygromycin phosphotransferase, xanthineguanine phosphoribosyl transferase, the Herpes simplex virus type 1 thymidine kinase, adenine phosphoribosyltransferase and hypoxanthine phosphoribosyltransferase.
  • the negative selectable marker may for example be selected from Herpes simplex virus type 1 thymidine kinase, adenine phosphoribosyltransferase, hygromycin phosphotransferase and hypoxanthine phosphoribosyltransferase.
  • the selectable markers are conveniently derived (e.g. by subcloning using restriction endonucleases) from any of a large number of known vectors, examples of which are described in e.g. Molecular Cloning: A laboratory Manual Second Edition Edited by Sambrook J, Fritsch and Maniatis T 1989 Cold Spring Harbour Laboratory Press).
  • expression elements for use in the invention may take any form so long as they can (under at least some circumstances) be made to direct and/or control the expression of the genes with which they are operably coupled.
  • Expression elements for use in the invention may comprise transcriptional and/or translational elements, and include promoters, ribosome binding sites, enhancers and regulatory sites including activator and repressor (operator) sites.
  • Preferred expression elements comprise promoters selected from a wide range available for use, examples of which are shown in Table 1. This Table, which is non-exhaustive, also indicates the use to which each promoter may be put in the methods of the invention described infra.
  • the expression elements for use in the invention may be selected from: promoters and/or enhancers which are specifically active in: (i) dopaminergic, serotoninergic, GABAergic, cholinergic or peptidergic neurones and sub-populations thereof; (ii) oligodendrocytes, astrocytes and sub-populations thereof; (iii) the endocrine glands, lungs, muscles, gonads, intestines, skeletal tissue or part or parts thereof; (iv) epithelial, fibroblast, fat, mast, mesenchymal or parenchymal cells; (v) particular stages of embryogenesis, and (vi) components of the blood system (e.g.
  • T-lymphocytes T-lymphocytes, B-lymphocytes and macrophages
  • they may be selected from promoters and/or enhancers which direct the transcription of genes for: (i) neurotransmitter-specific receptors; (ii) ion channels; (iii) receptors involved in ion channel gating and (iv) cytokines, growth factors and hormones.
  • At least one of the selectable markers may advantageously be constitutively expressed. This ensures uniform expression of the selectable marker in every transgenic cell of the transgenic organism under all conditions, which is particularly useful where the transgenic organism is for general use as a source organism for cell/tissue culture.
  • Serotonin 2 Glial cells in Neuro- receptor serotoninergic degenerative projection areas diseases Table 1 ( cont . .
  • CD4 receptor CD4 expressing AIDS
  • Constitutive expression may be achieved for example via the use of a promoter which directs the expression of a "house-keeping" gene.
  • a "house-keeping" gene is one which is expressed in all cell types. Their translated products are required as part of general cell metabolism or cell structure and, consequently, they are not specifically expressed in a particular cell or tissue type. House-keeping gene promoters, therefore, need to be active in a broad range of (and sometimes in all) cell types in order to ensure constitutive gene expression.
  • H-2K b class 1 promoter for the histocompatability complex H-2K b class 1 promoter (Weiss et al. (1983) Nature, 301, 671-674; Baldwin and Sharp (1987), Mol. Cell. Biol. 7, 305-313; Kimura et al. (1986), Cell 44, 261-272) which has been shown to express downstream coding sequences in cells generally when used as a promoter in a transgene (Jat et al (1991), PNAS USA 88, 5096-5100).
  • Another example is the viral SV40 early promoter.
  • the promoters for use in the present invention are not restricted to those derived from mammalian cells but may also include avian- and fish-derived promoters. Additionally, virally derived promoters, some of which have biological activity in a broad range of mammalian, fish and avian cells as well as other eukaryotes, could also be used in performing the invention. Examples are the simian virus-40 derived early or late promoters, or the Long Terminal Repeats (LTR'S) of retroviruses which comprise promoter as well as enhancer elements and have the ability to promote expression of sequences under their influence in a broad range of eukaryote cells.
  • LTR'S Long Terminal Repeats
  • promoters along with supporting sequences such as enhancer elements and other regulatory elements are well known to the man skilled in the art (see e.g. Molecular Cloning: A laboratory Manual Second Edition Edited by Sambrook J, Fritsch and Maniatis T 1989 Cold Spring Harbour Laboratory Press).
  • the transgenic organism of the invention may also contain heterologous DNA which further comprises a reporter transgene, for example 3-galactosidase or luciferase.
  • the reporter transgene may be itself operably linked to an expression element or elements which are subject to cell- or tissue-specific regulation.
  • Such reporter transgenes facilitate subsequent analysis of cells/tissue cultured from the transgenic organism and in particular permit the response (to for example an induced deficit in a particular class of cells/tissue) of a particular expression element or class of expression elements to be monitored in vivo or in vitro.
  • the invention provides a method of culturing cells and/or tissues in vitro, comprising the steps of: (a) providing a transgenic animal or plant having cells containing genetic material which confers a selectable phenotype thereon; (b) generating a primary culture from cells and/or tissue of the transgenic organism of step (a); and (c) selectively growing the primary culture on the basis of the selectable phenotype conferred by the genetic material contained in the cells of the transgenic organism.
  • the cell/tissue culture method of the invention is based on the use of a transgenic organism having a selectable marker operably linked to a tissue- or cell-specific expression element or elements, whereby in step (c) a particular cell/tissue type is selectively grown on the basis of the tissue- or cell-specific expression therein of said at least one selectable marker.
  • This preferred method of the invention finds application for example in the selection of thyroid follicular cells from a primary (mixed cell) culture.
  • This method may provide a primary stromal cell population of the thyroid gland in the absence of the thyroid follicular cells and constitutes a unique cell culture system useful for the study of thyroid biology and in the development of new therapeutic drugs for the treatment of thyroid diseases.
  • step (c) reduces or eliminates microbial contamination of the tissue culture, thereby alleviating or eliminating a common problem in cell culture systems, viz. culture contamination (particularly by fungi and yeasts ) .
  • transgenic organisms of the invention can also be used as a source of lymphocytes in methods for the production of monoclonal antibodies.
  • Monoclonal antibodies are of fundamental importance in biotechnology. Their preparation involves a sequence of steps including: (a) immunizing an animal by injecting the antigen of interest, (b) removing the spleen from the animal and preparing lymphocytes therefrom, (c) fusing the lymphocytes with immortal (usually myeloma) cells to produce a hybridoma, (d) selectively growing hybridomas and (e) cloning the hybridomas to produce a clone secreting the monoclonal antibody of interest.
  • immortal usually myeloma
  • step (d) The step of selectively growing the hybridoma (step (d) , above) is usually achieved on the basis of a HPRT" genotype in a myeloma fusion partner which prevents unfused myeloma cells from growing in selective media containing hypoxanthine, aminopeterin and thymidine (HAT medium). This restricts the choice of fusion partners.
  • a method of making a monoclonal antibody specific for an antigen comprising the steps of: (a) providing a transgenic animal (for example a rat, rabbit, pig or mouse) having lymphocytes containing genetic material which confers a selectable phenotype thereon; (b) immunizing the transgenic organism with the antigen; (c) removing the lymphocytes from the transgenic animal; (d) fusing the lymphocytes of step (c) with immortal cells (for example tumour cells, e.g. myeloma cells) to produce hybridomas; and (e) selectively culturing the hybridomas on the basis of the selectable phenotype conferred by the genetic material contained in the lymphocytes.
  • a transgenic animal for example a rat, rabbit, pig or mouse
  • immortal cells for example tumour cells, e.g. myeloma cells
  • the presence of the selectable marker in the lymphocyte preparations from the transgenic animal obviates the requirement for eg. a HPRT selection process and expands the repertoire of fusion partner cells that can be used in hybridoma formation.
  • transgenic organisms of the present invention also find application in relation to diseases or disorders involving cell loss.
  • neurodegenerative disorders such as Parkinson's disease, Huntington's chorea and Alzheimer's disease one or more sub-populations of neurotransmitter-identified cells are lost during the course of the disease.
  • Parkinson's disease this loss is principally of the dopaminergic neurones of the substantia nigra region of the brain, although other cell types also decline.
  • Alzheimer's disease there is a decrement in acetylcholine-, serotonin- and noradrenaline- containing neurones projecting to the neo- and palaeocortex.
  • neurological diseases and disorders also stem from neural cell degeneration; the demyelination occurring in multiple sclerosis, for instance, is due to the destruction of oligodendrocytes in the brain.
  • HIV Human Immunodeficiency Virus
  • SIV semavirus immunodeficiency virus
  • SIV monkeys do not acquire full-blown AIDS. In many instances, they show no symptoms at all.
  • Alternative models that have been proposed include HIV-infected chimpanzees. Apart from the potential ethical considerations, the manisfestation of AIDS-like symptoms in such a model may take several years, substantially hindering research and the development of effective therapies.
  • a method of selectively eliminating or depleting a particular tissue or cell type in an organism comprising the steps of: (a) providing a transgenic organism having a negative selectable marker operably linked to an expression element (e.g. a promoter) specific for the tissue or cell type to be eliminated or depleted, and (b) administering a selective agent to the organism to eliminate or deplete that tissue or cell type on the basis of the expression therein of the negative selectable marker.
  • an expression element e.g. a promoter
  • the selective agent may be administered by any route. Where systemic administration is required, oral, parenteral or intravenous routes may be used. Where localized administration is required (for example where the tissue or cell-type to be eliminated is restricted to a particular organ or to a particular region of the body) targeted injection, implantation (e.g. slow release capsules) or catheterization may be used. For example, tissue in particular regions of the brain may be specifically targeted by intracerebral injection.
  • the method of selectively eliminating or depleting a particular tissue or cell type of the invention may be employed to provide in vivo models of diseases/disorders involving disease-related cell loss, for example im unodegenerative or neurodegenerative diseases/disorders (such as AIDS, Parkinson's and Alzheimer's disease).
  • diseases/disorders involving disease-related cell loss for example im unodegenerative or neurodegenerative diseases/disorders (such as AIDS, Parkinson's and Alzheimer's disease).
  • the present invention provides a method of modelling disease/disorder-related cell/tissue loss or atrophy comprising the steps of: (a) providing a transgenic organism having a negative selectable marker operably linked to an expression element (e.g. a promoter) specific for the tissue or cell type which is subject to disease-related elimination or atrophy; and (b) administering a selective agent to the organism to eliminate or deplete the tissue or cell type on the basis of the expression therein of the negative selectable marker.
  • an expression element e.g. a promoter
  • the invention also provides a method (e.g. an in vitro method) of determining the effect of a deficit in a first class of cells on the characteristics of a second class of cells in an organism, the method comprising the steps of: (a) providing a transgenic organism having a first negative selectable marker operably linked to an expression element specific for the first class of cells and either; (i) a positive selectable marker operably linked to an expression element specific for the second class of cells, or (ii) a second negative selectable marker linked to an expression element which directs the expression of the negative selectable marker in all cells of the organism except the second class of cells; (b) administering a selective agent to the organism to induce a deficit in the first class of cells on the basis of the expression therein of the negative selectable marker; (c) removing cells from the organism; and (d) selectively culturing cells of the second class from those cells removed in step (c) on the basis of; (i) the expression therein of the positive selectable marker, or (ii) the lack of
  • the invention provides a method of screening compounds for pharmacological activity against a disease or disorder involving cell/tissue loss or atrophy, comprising the steps of: (a) providing a test model of the disease via the steps of; (i) providing a transgenic organism having a negative selectable marker operably linked to an expression element (e.g.
  • a promoter specific for the tissue or cell type which is subject to disease/disorder-related elimination or atrophy, and then (ii) administering a selective agent to the organism to eliminate or deplete the tissue or cell type on the basis of the expression therein of the negative selectable marker to produce a test model; (b) administering the compound to be tested to the test model; (c) screening the compound to be tested on the basis of its effect on the test model of step (a) .
  • the methods of the invention may be usefully applied to any disease or disorder which is associated with cell/tissue loss or atrophy.
  • the methods of the invention find particular utility in respect of neurodegenrative or immunodegenerative diseases and disorders, for example: (a) Parkinson's disease (the tissue or cell-type to be eliminated or depleted comprising dopaminergic neurones in the substantia nigra); (b) Huntington's chorea (the tissue or cell-type to be eliminated or depleted comprising neural cells of the striatum; (c) Alzheimer's disease (the tissue or cell-type to be eliminated or depleted comprising acetylcholine-, serotonin- and/or noradrenaline- neurones associated with the neo- and palaeocortex; (d) multiple sclerosis (the tissue or cell-type to be eliminated or depleted comprising brain oligodendrocytes) , (e) immune disease and the cell-type to be eliminated or depleted co prises CD3, CD4
  • the method of the invention could be used to specifically deplete or eliminate CD4 cells by linking a negative selectable marker to a CD4 cell-specific promoter (e.g. the CD4 receptor promoter). This would permit the generation of an in vivo model of AIDS by regulating the proportion of cells expressing CD4 by negative selection in vivo.
  • a CD4 cell-specific promoter e.g. the CD4 receptor promoter
  • any residual CD4 expressing cells could later be isolated from the transgenic tissue of the animal model by positive selection in vitro for further study.
  • the invention also contemplates cell/tissue cultures derived from the transgenic organisms of the invention (or produced by the cell culturing methods of the invention) , and also to various therapeutic uses of the invention.
  • Thyroglobulin-thvmidine kinase-internal ribosomal entry site-neomvcin resistance TG-TK- ⁇ -IRES-neo r .
  • neomycin resistance gene (neo r ) was obtained from the pBabe Neo plasmid (Morgenstern & Land, Nucl. Acids Res. 18(1990)3587-3596) by digestion with Hind Ill/Cla I and retrieval of the 1165 b.p. fragment containing the neo r gene by gel electrophoresis and the Promega Wizard PCR kit.
  • the pPBS plasmid (Morgan, Nucl. Acids Res. (1992) 20, pages 1293-1299) comprising the poliovirus derived internal ribosomal entry site sequence was digested with Hind Ill/Cla I. However, this could not be done simultaneously, or, in sequence, since the restriction sites were too close together. In order to overcome this problem, the plasmid was initially digested with Hind III and a 200 b.p. fragment of DNA containing Hind III restriction sites at both the 5' and 3' ends was inserted in order to separate the sites. The pPBS plasmid could then be digested first with Cla I and then with Hind III.
  • Terminal phosphate groups were removed from the Hind III/Cla I cut pPBS vector using calf intestinal phosphatase (CIP).
  • CIP calf intestinal phosphatase
  • the neomycin gene was then ligated into the pPBS plasmid overnight at 15°C and the ligation reaction transformed into freshly-made MC1061 competent cells.
  • Positive colonies were identified by digestion of prepared plasmids with Hind Ill/Cla I. The neo r gene and plasmid being detected electrophoretically in plasmid preparations from positive colonies. Plasmids from the positive colonies were then digested with Hinc II and Sac I (both restriction enzymes leaving digested DNA with blunt ends). The resulting Sac I/Hinc II digestion containing the IRES-neo R fragment was run on a 1% electrophoresis gel and the appropriate size band was excised and the DNA electroeluted and ethanol- precipitated.
  • the TG-TK ⁇ plasmid (freely available from Genbank, NIH, USA accession No. J02224, Santelli et al 1993) DNA was prepared using Promega Wizard mini preps and digested with Nar I. The ends of the plasmid were blunted using T4 Polymerase at 37 ⁇ C for 1 h followed by removal of the terminal phosphate groups using CIP. The CIP was inactivated by treatment of the DNA with phenol/chloroform followed by ethanol precipitation. The resulting plasmid was electrophoresed on a 1% agarose gel and the DNA was recovered and ligated with the insert in a 1:3 molar ratio of plasmid to insert.
  • the ligation was incubated at 15°C overnight, and was then used to transform competent MC1061 cells. Positive colonies were selected by digestion of prepared plasmids with BamH I (the correct construct provided restriction fragments of size 3980, 1663, 3102 and 1039 b.p.).
  • pCD2 plasmid (Mullen et al., PNAS 89(1992)33-37) was digested with EcoR I and EcoR V, and the digest was electrophoresed on a 1% agarose gel where the 2.5 kb. fragment containing the cytosine deaminase gene, the SV40 promoter and the neomycin resistance gene was retrieved by electroelution followed by ethanol preciptation.
  • the pCI vector was digested with EcoR 1 and S a I (a restriction enzyme leaving the DNA with blunt ends) , and the terminal phosphate groups were removed using CIP and the enzyme was inactivated with phenol/chloroform followed by ethanol precipitation. The band was then gel-purified and recovered by electroelution.
  • the ligation was set up containing a 3:1 molar ratio of insert to vector and was carried out at 15°C overnight.
  • the ligation mixture was used to transform freshly- prepared MC1061 competent cells and positive colonies were selected by digestion of prepared plasmids with EcoR 1 and Hind III to provide restriction fragments of length 1868 b.p. and 5062 b.p., respectively. Linearization of the plasmid was achieved by digestion with Bgl I.
  • Transgenic rats were produced by established methods (Hogan, B., Constantini, F. & Lacy, E. (1986)
  • Fibroblast cultures derived from lung of adult CD2/neo r , TG/TK/neo r and control animals were produced and expanded by routine methods (Freshney (1987), Alan R. Liss, New York) . Twenty-four hours after plating, geneticin (400 ⁇ g/ml) was added to cultures originating from both types of transgenic rats and from control rats, and replaced every three days with fresh medium. When required, cells were subcultured (1:3) to prevent them becoming confluent, again by basic culture methods (Freshney 1987). Cell counts were made manually in 20 fields chosen randomly and the values at each time point, after allowing for changes due to subculturing, were aggregated.
  • 5-fluorocytosine The effects of 5-fluorocytosine (5FC) were also determined. 5-fluorocytosine at a concentration of lOO ⁇ g/ml had no effect on fibroblast cells derived from control animals or from the TG/TK/neo r transgenic. In the cells derived from the CD2/neo r transgenic animal, however, 94% of the originally-plated cells died, or were non-functional (as determined by their failure to exclude trypan blue) after 10 days culture in the presence of 5FC (Table 2). By contrast, no significant difference in cell counts was found between cultures from control rats in the absence and presence of 5FC, or between controls and cultures taken from CD2/neo r rats in the absence of added 5FC (Table 2) .
  • Table 2 Survival of lung fibroblast cells derived from control and transgenic rats, and effects of various drugs.
  • Thyroid cultures derived from the thyroid gland of adult CD2/neo r , TG/TK/neo r and control animals were produced by routine methods (Freshney, 1987). Twenty-four hours after plating, geneticin (400 ⁇ g/ml) was added to cultures originating from both types of transgenic and the control rats, and replaced every three days with fresh medium. When required, cells were subcultured (1:2) to prevent their becoming confluent. Cell counts were made manually in 20 fields chosen randomly, and the values at each time point, after allowing for changes due to subculturing, were aggregated (Table 3).
  • Table 3 Survival of thyroid cells derived from control and transgenic rats, and effects of various drugs.
  • Drugs were added at day 2 in culture. Values are related to the number of cells found in control cultures without drug additions at various times after plating, and allow for dilutions resulting from passaging. Figures are the means of three separate determinations, the standard errors all being less than 15% of the mean. E. Enhanced sterility in tissue culture using cells from CD2-neo r transgenic animals
  • Fig. 3 shows phase- contrast photomicrographs of 60 days in vitro spinal cord cultures derived from control rat embryos (A) and from CD2/neo r transgenic rat embryos (B) at day 14 of gestation.
  • the latter cultures were deliberately infected with yeast and other microorganisms, and simultaneously treated with geneticin. No infection could be discovered after geneticin application, whereas infected cultures without geneticin were overrun with microorganism growth within 4 or 5 days).
  • both types of transgenic animal, or the cells therefrom were apparently normal until application of either ACG or 5FC, as appropriate. After such application, either in vivo or in vitro, the cells upon which sensitivity had been conferred were rapidly destroyed. In addition, cells from both transgenic animals were resistant to the cytotoxic effects of geneticin, whereas cells from non-transgenic controls were completely eradicated.
  • Example 2 Proposed protocol for the production of a transgenic mouse bearing both positive and negative selectable markers
  • HSV herpes simplex virus
  • tk thymidine kinase gene
  • neo bacterial neomycin phosphotransferase gene
  • the plasmid vector is digested with restriction endonucleases and a fragment containing both the tk and neo selectable markers (along with the expression elements operably linked thereto) is isolated on an agarose gel.
  • the fragment isolated on the gel is then purified and injected into male pronuclei of fertilized one-cell mouse eggs at a concentration of 1-2 ug/ml DNA in TE buffer (lO M Tris, Ph 7.5, 0.2 Mm EDTA) .
  • the eggs are those derived from a CBA x C57BL/10 mating.
  • the eggs which survive micro-injection are then transferred to pseudopregnant females as described e.g. in Wagner et al. (1981) PNAS 78, 5016, and allowed to develop to term.
  • each pup is analysed to determine whether the transgenes are present.
  • DNA is prepared from a section of the tail by the method described in Sambrook et al. (1989) "Molecular Cloning", Cold Spring Harbor. The presence of the neo and tk genes is determined by probing with labelled tk and neo-specific probes.
  • transgenic pups so identified are mated and their offspring also analysed to check for Mendelian transfer of the transgenes.
  • Example 3 Proposed protocol for the selective culture of mouse thyroid follicular cells
  • Transgenic mice are prepared as described in Example 1, except that the neo gene is placed under the control of a thyroglobulin promoter (e.g. described by Christophe et al. (1989) Molecular and cellular endocrinology 64(1) 5- 18; Christophe et al (1987), 19, Suppl. 17, pp 70-73; and Ledent et al. (1990), PNAS, 87 (16), pp 6176-6180).
  • a thyroglobulin promoter e.g. described by Christophe et al. (1989) Molecular and cellular endocrinology 64(1) 5- 18; Christophe et al (1987), 19, Suppl. 17, pp 70-73; and Ledent et al. (1990), PNAS, 87 (16), pp 6176-6180).
  • the transgenic mice are sacrificed and the thyroid tissue removed and a primary culture prepared in the presence of antibiotic G418. This antibiotic kills cells not expressing the neo gene, and results in the selective culturing within the primary (mixed cell) culture of thyroid follicular cells.
  • the bacterial neomycin phosphotransferase (neo) gene (operably linked to the SV40 early promoter) is cloned into the appropriate cloning site of a plasmid vector.
  • the plasmid is then digested with restriction endonucleases and a fragment containing the neo selectable marker is isolated on an agarose gel, and transgenic mice bearing the neo transgene are then prepared essentially as described in Example 1.
  • the antigen against which a monoclonal antibody is required is purified and injected into the transgenic mouse prepared as described above along with Freund's adjuvant.
  • the mouse is then sacrificed and the spleen removed and placed in tissue culture fluid.
  • the spleen is teased apart to release the lymphocytes and these are isolated by centrifugation.
  • the lymphocytes are then mixed with a myeloma fusion partner in the presence of polyethylene glycol to induce fusion and produce hybridomas.
  • Hybridomas are selected by supplementing the culture medium with the antibiotic G418 on the basis of the presence of the neo selectable marker in the mouse lymphocytes.
  • the hybridomas are then cloned by limiting dilution and the relevant clone identified by screening via the appropriate binding assay.
  • the myeloma cell line does not need to have a negative selectable marker (e.g. HPRT"). Moreover, the presence of G418 in the culture medium reduces or eliminates the risk of culture infection.
  • a negative selectable marker e.g. HPRT
  • Example 5 Proposed protocol for the preparation of a rattine model of Parkinson's disease
  • the herpes simplex virus (HSV) thymidine kinase gene (tk) is operably linked to a promoter which is active only in dopaminergic neurones in the substantia nigra and cloned into the appropriate cloning site of a plasmid vector.
  • HSV herpes simplex virus
  • tk thymidine kinase gene
  • the plasmid is digested with a restriction endonuclease and a fragment containing the tk selectable marker is isolated on an agarose gel, and transgenic rats bearing the tk transgene are then prepared essentially as described in Example 1.
  • Ganciclovir is then administered by injection into the substantia nigra regions of the brain of the transgenic rats to specifically eliminate or deplete the dopaminergic neurones expressing the negative selectable tk marker, thus providing a rattine model of Parkinson's disease.
  • Example 6 Proposed protocol for the preparation of a rattine model of Alzheimer's disease
  • HSV herpes simplex virus
  • tk thymidine kinase gene
  • the plasmid is digested with a restriction endonuclease and a fragment containing the tk selectable marker is isolated on an agarose gel, and transgenic rats bearing the tk transgene are then prepared essentially as described in Example 1.
  • Ganciclovir is then administered by injection into the aapropriate region of the brains of the transgenic rats to specifically eliminate or deplete the acetylcholine-, serotonin- and/or noradrenaline- neurones associated with the neo- and palaeocortex expressing the negative selectable tk marker, thus providing a rattine model of Alzheimer's disease.

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Abstract

Organismes transgéniques (en particulier animaux ou plantes transgéniques) portant des marqueurs positifs et/ou négatifs pouvant être sélectionnés, et utilisation desdits organismes dans divers procédés, dont des techniques de culture tissulaire/cellulaire, des procédés de préparation d'anticorps monoclonaux, des procédés d'élimination ou d'appauvrissement sélectif d'un type tissulaire/cellulaire particulier, des procédés de triage de composés selon leur activité pharmacologique et des procédés permettant de déterminer l'effet d'un déficit dans une première catégorie de cellules ou les caractéristiques d'une seconde catégorie de cellules dans un organisme.
EP95936026A 1994-11-03 1995-11-03 Organismes transgeniques et leurs utilisations Withdrawn EP0789757A1 (fr)

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AL-SHAWI R. ET AL: "Sexual Dimorphism and Growth Hormone Regulation of a Hybrid Gene in transgenic Mice", MOLECULAR ENDOCRINOLOGY, vol. 6, 1992, pages 181 - 190 *
BORRELLI E.: "Targeting of an inducible phenotype in animal cells", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 85, October 1988 (1988-10-01), U.S.A., pages 7572 - 7576 *
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