DE10041842A1 - Genetically modified, non-human mammal that contains an additional inducible regulator gene - Google Patents
Genetically modified, non-human mammal that contains an additional inducible regulator geneInfo
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- DE10041842A1 DE10041842A1 DE2000141842 DE10041842A DE10041842A1 DE 10041842 A1 DE10041842 A1 DE 10041842A1 DE 2000141842 DE2000141842 DE 2000141842 DE 10041842 A DE10041842 A DE 10041842A DE 10041842 A1 DE10041842 A1 DE 10041842A1
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Classifications
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- 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
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- 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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- 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)
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- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
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Abstract
Description
Die Erfindung betrifft ein transgenes, nicht-menschliches Säugetier, vorzugsweise eine Maus, dessen Körper- und Geschlechtszellen ein induzierbares Regulatorgen enthalten, welches in allen Zellen exprimiert wird. Das Genprodukt des Regulatorgens supprimiert die Aktivität von NF-κB Transkriptionsfaktoren. Vorzugsweise kodiert das Regulatorgen eine mutierte Form des natürlichen NF-κB Inhibitors IκBα, der aminoterminale Degradationssignale fehlen. Dieses IκBα Mutanten-Protein, IκBαΔN, bindet an zelluläre NF-κB Proteine und hält diese in einem inaktiven Zustand. Im Gegensatz zu dem natürlichen IκBα, welches nach zellulärer Stimulierung mit einer Vielzahl unterschiedlicher NF-κB aktivierender Signalstoffe degradiert wird und NF-κB dadurch freisetzt, bleibt IκBαΔN stabil und blockiert damit eine NF-κB Freisetzung.The invention relates to a transgenic, non-human mammal, preferably one Mouse, whose body and sex cells contain an inducible regulator gene, which is expressed in all cells. The gene product of the regulatory gene suppresses the Activity of NF-κB transcription factors. The regulator gene preferably encodes one mutated form of the natural NF-κB inhibitor IκBα, the amino terminal Degradation signals are missing. This IκBα mutant protein, IκBαΔN, binds to cellular NF-κB proteins and keeps them in an inactive state. In contrast to the natural IκBα, which after cellular stimulation with a variety of different NF-κB activating signaling substances is degraded and NF-κB is released, IκBαΔN remains stable and thus blocks NF-κB release.
Das genetisch veränderte Säugetier wird durch eine Kombination der homologen Rekombinationstechnik und des Cre-Lox Systems erhalten. Bei der Cre-Lox-Methode wird die Cre Rekombinase des P1 Bakteriophagen verwendet, die an zwei benachbarte spezifische kurze DNA Sequenzen, den loxP Stellen, bindet. Nach Bindung an die loxP Sequenzen spaltet Cre beide Sequenzen nukleolytisch und fügt die Enden unter Verlust der dazwischen liegenden DNA Sequenzen wieder zusammen.The genetically modified mammal is made by a combination of the homologous Recombination technology and the Cre-Lox system preserved. The Cre-Lox method is the Cre recombinase of P1 bacteriophage used to target two neighboring specific binds short DNA sequences, the loxP sites. Cleaves after binding to the loxP sequences Cre nucleotide both sequences and inserts the ends losing the one in between DNA sequences together again.
Homologe Rekombinations-Technologien sind an sich bekannt und sind z. B. in Hogan et al., 1994, beschrieben. Die Cre-Lox Technologie zur Erzeugung konditionaler Geninaktivierung ist ebenfalls an sich bekannt und als Methode im Detail erläutert (Gu et al., 1994; DiSanto et al., 1995; Schwenk et al., 1995). Des weiteren liegen bereits zahlreiche Patente zur Herstellung transgener, nicht-menschlicher Säugetiere vor, wie z. B. die sogenannte Krebsmaus (US-PS 4,736,866).Homologous recombination technologies are known per se and are e.g. B. in Hogan et al., 1994. The Cre-Lox technology for the generation of conditional Gene inactivation is also known per se and is explained in detail as a method (Gu et al., 1994; DiSanto et al., 1995; Schwenk et al., 1995). Furthermore, there are already numerous Patents for the production of transgenic, non-human mammals, such as. B. the so-called crab mouse (US-PS 4,736,866).
Die biologische Aktivität des NF-κB Regulatorgens wird nur nach Kreuzung mit einem Rekombinase (Cre) - exprimierenden Tier erhalten. Das Regulatorgen enthält zur Gewährleistung seiner Cre-abhängigen Expression ein von loxP Elementen flankiertes Stop Kodon vor der Translations-Startstelle. Ein translatierbares Transkript kann also nur nach Cre-vermittelter Rekombination erhalten werden. Das Regulatorgen wurde dem Ursprungstier durch Transfektion und Rekombination embryonaler Stammzellen und deren Injektion in Blastozysten übertragen. Anwendungsgebiete der erfindungsgemäßen Tiere liegen in der medizinischen, der pharmazeutischen und der biologischen Grundlagen- und anwendungsorientierten Forschung und Diagnose.The biological activity of the NF-κB regulatory gene only becomes apparent after crossing with an animal expressing recombinase (Cre). The regulatory gene contains Ensuring its Cre-dependent expression a stop flanked by loxP elements Codon in front of the translation start point. A translatable transcript can only be used after Cre-mediated recombination can be obtained. The regulatory gene became the original animal by transfection and recombination of embryonic stem cells and their injection into Blastocysts transmitted. Areas of application of the animals according to the invention are in the medical, pharmaceutical and biological basic and application-oriented research and diagnosis.
Genetisch veränderte Tiere lassen sich durch den Prozess homologer Rekombination in embryonalen Stammzellen (ES), gefolgt von Injektion der so modifizierten Stammzellen in Blastozysten, erzeugen. Bei der homologen Rekombination wird ein normales Gen durch ein künstlich verändertes Gen durch den Rekombinationsvorgang ersetzt. Häufig wird diese Methode angewendet, um Gene durch die Rekombination völlig zu inaktivieren und deren Funktion nachfolgend zu bestimmen. Hierbei wird in der Regel ein Targetingkonstrukt verwendet, welches den Leserahmen des Gens entweder durch Einfügen eines vorzeitigen Stopp-Kodons oder durch Ersetzen der kodierenden exonischen Sequenz durch ein Markerprotein zerstört. Homologe Rekombination wird jedoch auch verwendet, um gezielt Punktmutationen in ein endogenes Gen einzufügen, wobei der im Targetingkonstrukt enthaltende Gen-Anteil eben nur in einer spezifischen Position verändert wird. Zunächst werden nach Transplantation der mit rekombinanten ES Zellen mikroinjizierten Blastozysten in Muttertiere chimäre Nachkommen erhalten. Diese Nachkommen tragen die eingefügte Gen-Veränderung nur auf einem der beiden Allele. Homozygote Tiere werden durch weitere Paarung erhalten.Genetically modified animals can be made through the process of homologous recombination in embryonic stem cells (ES), followed by injection of the modified stem cells into Blastocysts. In homologous recombination, a normal gene is replaced by a artificially modified gene replaced by the recombination process. This is common Method used to completely inactivate genes by recombination and their Function to be determined below. This is usually a targeting construct which uses the reading frame of the gene either by inserting a premature one Stop codons or by replacing the coding exonic sequence with a Marker protein destroyed. However, homologous recombination is also used to target Insert point mutations into an endogenous gene, the one in the targeting construct containing gene portion is only changed in a specific position. First after transplantation of the blastocysts microinjected with recombinant ES cells Chimeric offspring obtained in dams. These descendants carry the inserted Gene change only on one of the two alleles. Homozygous animals are identified by others Get pairing.
Homologe Rekombination wird im Rahmen dieser Erfindung eingesetzt, um ein künstlich verändertes Gen (vorzugsweise IκBαΔN) in ein Allel eines zellulären Genes (hier β-Catenin) so zu inserieren, daß es die gleiche ubiquitäre Expression wie das zelluläre Gen (z. B. β-Catenin) erlangt, den Leserahmen des zellulären Gens jedoch zerstört. Die Tiere werden nur heterozygot gehalten, da homozygote Inaktivierung des endogenen Gens (z. B. β-Catenin) letal ist (Haegel et al., 1995, Hülsken et al., 2000). Die Expression des veränderten Genlokus ist auf mRNA Ebene ständig gewährleistet, die mRNA kann jedoch wegen der eingefügten loxP-Stopp-loxP Kassette nicht in ein funktionelles Protein übersetzt werden. Daher bleiben die Tiere frei von Symptomen und sind nach phänotypischen Merkmalen von den Wildtyp-Tieren nicht zu unterscheiden.Homologous recombination is used in the context of this invention in order to artificially modified gene (preferably IκBαΔN) into an allele of a cellular gene (here β-catenin) so that it has the same ubiquitous expression as the cellular gene (e.g. β-catenin), but destroys the reading frame of the cellular gene. The animals are only kept heterozygous because homozygous inactivation of the endogenous gene (e.g. β-catenin) is lethal (Haegel et al., 1995, Hülsken et al., 2000). The expression of the changed Gene locus is constantly guaranteed at the mRNA level, however, the mRNA can because of the inserted loxP-stop-loxP cassette cannot be translated into a functional protein. Therefore, the animals remain free of symptoms and are of phenotypic characteristics indistinguishable from the wild-type animals.
Um das künstlich veränderte Gen in eine biologisch aktive Form zu bringen, muß das Tier mit einem anderen Tier gekreuzt werden, welches ein transgenes Tier ist und die Cre- Rekombinase als zusätzliches Gen enthält. Dieses zweite Tier kann die Cre-Rekombinase entweder unter Kontrolle eines ubiquitär aktiven Promoters exprimieren, oder unter Kontrolle eines gewebs- oder zellspezifischen Promoters. Die Cre-Rekombinase kann weiterhin durch einen künstlich induzierbaren Promoter reguliert sein. Hierfür stehen Tetrazyklin-regulierte Promotoren zur Verfügung. Weiterhin stehen Cre-Rekombinase-Fusionen mit Steroidbindungs-Domänen von Östrogen oder Progesteron-Rezeptoren zur Verfügung, oder werden entwickelt, die durch synthetische Steroidderivate aktiviert werden (z. B. Tamoxifen oder Progesteron-Derivate). Hier führt der Steroidrezeptor-Fusionsanteil zu einer Repression der enzymatischen Aktivität von Cre, die nach Steroidbindung aufgehoben wird.In order to bring the artificially modified gene into a biologically active form, the animal has to be crossed with another animal, which is a transgenic animal and the Cre- Contains recombinase as an additional gene. This second animal can be the Cre recombinase express either under the control of a ubiquitously active promoter, or under control a tissue or cell specific promoter. The Cre recombinase can continue to regulated an artificially inducible promoter. This is what tetracycline regulated stands for Promoters available. Cre-recombinase fusions are also available Steroid binding domains available from estrogen or progesterone receptors, or are being developed that are activated by synthetic steroid derivatives (e.g. tamoxifen or progesterone derivatives). Here the steroid receptor fusion portion leads to repression the enzymatic activity of Cre, which is released after steroid binding.
Die aus der Paarung von loxPIκBαΔN Tieren und Cre Mäusen erhaltenen Nachkommen exprimieren dann das künstlich veränderte Gen (vorzugsweise IκBαΔN), welches durch Cre aktiviert wurde, in biologisch aktiver Form entweder ubiquitär, oder in bestimmten Zelltypen, Geweben oder Organen. In diesen Fällen ist die Expression während der Embryonalentwicklung bereits gewährleistet und dauert über die gesamte Lebensspanne des Tiers an. Im Fall der induzierbaren Cre-Rekombinasen wird das künstlich veränderte Gen in biologisch inaktiver Weise gehalten, bis durch Zugabe des induzierenden Agens (z. B. Tetrazyklin, Tamoxifen, oder Progesteron-Derivate) Cre dereprimiert wird. Hierbei wird ermöglicht, das künstlich veränderte Gen zu einem gewünschten Zeitpunkt in biologisch aktiver Form zu exprimieren. Die Induktion kann entweder zu einem Zeitpunkt während der Embryonalentwicklung oder im erwachsenen Tier erfolgen.Those obtained from the pairing of loxPIκBαΔN animals and Cre mice Offspring then express the artificially modified gene (preferably IκBαΔN), which was activated by Cre, either biologically active in ubiquitous form, or in certain cell types, tissues or organs. In these cases, the expression is during the embryonic development is already guaranteed and lasts over the entire lifespan of the animal. In the case of the inducible Cre recombinases, the artificially modified gene kept in a biologically inactive manner until by adding the inducing agent (e.g. Tetracycline, tamoxifen, or progesterone derivatives) Cre is depressed. Here will enables the artificially modified gene at a desired time in biological to express active form. Induction can occur either at a time during the Embryonic development or in the adult animal.
Der Erfindung lag die Aufgabe zugrunde, ein genetisch verändertes Tier zur Verfügung zu stellen, welches geeignet ist, detaillierte Untersuchungen zur Pathogenese von Erkrankungen zu erlauben, bei denen die Transkriptionsfaktoren der NF-κB Multigen-Familie beteiligt sind. Diese Erkrankungen schließen ein weites Spektrum von Autoimmun- und Entzündungserkrankungen ein, wie Organabstoßung, multiple Sklerose, rheumatoide Arthritis, Psoriasis und andere Dermatosen, Asthma, Morbus Crohn und Artherosclerose. NF-κB ist weiter involviert bei septischem Schock, Infektion mit Parasiten und Bakterien und bei der Replikation pathogener Viren, wie z. B. HIV (Wulczyn et al., 1996; Barnes and Karin, 1997; Hatada et al. 2000). Wegen seiner anti-apoptotischen sowie zellteilungs-aktivierenden Wirkungen erlangt NF-κB bei der Resistenz von Tumoren gegen Chemo- oder Radiotherapie als mögliches therapeutisches Target eine generelle Bedeutung (Wang et al., 1999). NF-κB ist zudem bei der Pathogenese bestimmter Lymphome (z. B. Morbus Hodgkin) und Leukämien als Onkogen wirksam (Mayo and Baldwin, 2000). Es ist anzunehmen, daß bei noch weiteren Erkrankungen verschiedenster Art eine wichtige Funktion von NF-κB nachgewiesen wird. Die Zur-Verfügung-Stellung des genetisch veränderten Tiermodells soll die Entwicklung und Testung von Pharmaka und diagnostischen Mitteln aller Art ermöglichen und erleichtern. Von der Erfindung sollen neue Impulse für die Untersuchung und Therapie NF-κB assoziierter Erkrankungen aller Art ausgehen.The invention was based on the object of a genetically modified animal To provide, which is suitable, detailed studies on the pathogenesis of Allow diseases where the transcription factors of the NF-κB multigen family involved. These diseases include a wide range of autoimmune and Inflammatory diseases such as organ rejection, multiple sclerosis, rheumatoid Arthritis, psoriasis and other dermatoses, asthma, Crohn's disease and artherosclerosis. NF-κB is also involved in septic shock, parasite and bacterial infection and the replication of pathogenic viruses, such as. B. HIV (Wulczyn et al., 1996; Barnes and Karin, 1997; Hatada et al. 2000). Because of its anti-apoptotic as well as cell division activating NF-κB has effects on the resistance of tumors to chemotherapy or radiotherapy of general importance as a possible therapeutic target (Wang et al., 1999). NF-κB is also in the pathogenesis of certain lymphomas (e.g. Hodgkin's disease) and leukemia effective as an oncogene (Mayo and Baldwin, 2000). It can be assumed that with others Diseases of various kinds an important function of NF-κB is proven. The provision of the genetically modified animal model is intended to develop and Enable and facilitate testing of all kinds of pharmaceuticals and diagnostic agents. Of The invention is intended to provide new impulses for the investigation and therapy associated with NF-κB All illnesses go out.
Die Aufgabe wird mit einem genetisch veränderten, nicht-menschlichen Säugetier, insbesondere einem Kleinsäuger, wie Maus oder Ratte, erzielt, welche ein zusätzliches NF-κB Regulator-Gen trägt. Anwendungsgebiete der erfindungsgemäßen Tiere liegen also in der medizinischen, pharmazeutischen und biologischen Grundlagenforschung, sowie in anwendungsorientierter Forschung und Diagnose.The task is done with a genetically modified, non-human mammal, especially a small mammal, such as a mouse or rat, which has an additional NF-κB Regulatory gene carries. Areas of application of the animals according to the invention are therefore in the basic medical, pharmaceutical and biological research, as well as in application-oriented research and diagnosis.
Mit der Verwendung der erfindungsgemäßen Tiere können bislang durchgeführte Tierversuche an höheren Tieren oder an Freiwilligen wesentlich eingeschränkt werden. Die erfindungsgemäßen Tiere werden für die Neu- und Weiterentwicklung therapeutischer und diagnostischer Methoden und zur Prüfung der Verwendungsfähigkeit von Arzneimitteln zur Therapie und Diagnostik NF-κB assoziierter Erkrankungen dienen. Insbesondere haben die erfindungsgemäßen Tiere einen hohen Wert für Bestimmung der therapeutischen Nützlichkeit NF-κB reprimierender Substanzen (Target-Validierung). Es können für beliebige Verabreichungsmodalitäten (systemisch, Organ/Gewebe-limitiert, während der Embryogenese oder im erwachsenen Organismus) und Einwirkungszeiten potentieller NF-κB inhibierender Substanzen die zu erwartenden physiologischen Nebenwirkungen bestimmt werden.With the use of the animals according to the invention previously carried out Animal experiments on higher animals or on volunteers are significantly restricted. The Animals according to the invention are therapeutic and for the new and further development diagnostic methods and to test the usability of drugs for Therapy and diagnosis of NF-κB associated diseases. In particular, they have animals according to the invention have a high value for determining the therapeutic usefulness NF-κB repressing substances (target validation). It can be used for any Administration modalities (systemic, organ / tissue limited, during embryogenesis or in the adult organism) and exposure times of potential NF-κB inhibitors Substances, the expected physiological side effects are determined.
Anschließend wird die Erfindung näher erläutert.The invention is then explained in more detail.
Die IκBαΔN cDNA mit den kodierenden Sequenzen für Aminosäuren 71-317 von IκBα (Krappmann, 1996) wurde im korrekten Leserahmen in den β-Catenin Targeting Vektor (Huelsken, 2000) kloniert. Für loxP-IκBαΔN wurden zwei loxP Sequenzen, Bindungsstellen für die Cre Rekombinase, in den Leserahmen mit dem Startkodon des β-Catenin Gens kloniert (Abb. 1A). Die loxP Sequenzen flankieren ein Stopkodon, welches eine Expression von translatierbarer IκBαΔN mRNA verhindert. loxP-IκBαΔN Mäuse werden mit Transgenen Cre Tieren gepaart. Die loxP Sequenzen stammen aus dem Plasmid pAMA (Zhang, 1996). Heterozygote β-catw.t./β-catI κ B αΔ N (oder β-catw.t./β-catloxP Δ N) 129/OLA ES Zellen wurden durch Elektroporation hergestellt. Zwei unabhängige heterozygote ES Zellklone eines jeden Targeting Konstruktes wurden jeweils für Blastozysten-Injektionen zur Erzeugung chimärer Mäuse verwendet. Heterozygote Tiere wurden durch Züchtung mit C57B1/6 Mäusen erhalten. Tiere mit ausreichend hohem C57B1/6 Hintergrund wurden für weitere Untersuchungen verwendet. Nach der Geburt waren die IκBαΔN Tiere sofort phänotypisch erkennbar. loxPIκBαΔN Mäuse wurden durch PCR identifiziert.The IκBαΔN cDNA with the coding sequences for amino acids 71-317 from IκBα (Krappmann, 1996) was cloned in the correct reading frame into the β-catenin targeting vector (Huelsken, 2000). For loxP-IκBαΔN, two loxP sequences, binding sites for the Cre recombinase, were cloned into the reading frame with the start codon of the β-catenin gene ( Fig. 1A). The loxP sequences flank a stop codon, which prevents expression of translatable IκBαΔN mRNA. loxP-IκBαΔN mice are paired with transgenic Cre animals. The loxP sequences come from the plasmid pAMA (Zhang, 1996). Heterozygous β-cat wt / β-cat I κ B αΔ N (or β-cat wt / β-cat loxP Δ N ) 129 / OLA ES cells were produced by electroporation. Two independent heterozygous ES cell clones of each targeting construct were used for blastocyst injections to create chimeric mice. Heterozygous animals were obtained by breeding with C57B1 / 6 mice. Animals with a sufficiently high C57B1 / 6 background were used for further studies. After birth, the IκBαΔN animals were immediately phenotypically recognizable. loxPIκBαΔN mice were identified by PCR.
Durch homologe Rekombination erhaltene ES Zellklone, die gemäß Southern Blot Analysen IκBαΔN- oder loxPIκBαΔN-positiv waren, wurden in Western Blot Analysen auf IκBα und IκBαΔN Protein-Expression untersucht (Abb. 1B). Wie erwartet, exprimieren nur IκBαΔN-positive ES Zellen das IκBαΔN Protein. loxPIκBαΔN positive ES Zellen exprimieren kein IκBαΔN, da keine Cre-vermittelte Rekombination erfolgte. Die Effizienz der IκBαΔN-vermittelten Inhibition der NF-κB DNA Bindungsaktivität wurde in Gel- Retardierungs-Assays untersucht (Abb. 1C). Während NF-κB p50-p65 Bindungsaktivität in loxPIκBαΔN positiven ES Zellen durch PMA induziert werden konnte, konnte keine induzierte Bindungsaktivität in IκBαΔN positiven ES Zellen festgestellt werden (Abb. 1C). IκBαΔN- und loxPIκBαΔN ES Zellklone wurden dann verwendet, um chimäre Mäuse durch Blastozysten-Injektion zu erzeugen. Heterozygote Mäuse wurden durch nachfolgendes Kreuzen der Chimären mit C57B1/6 Wildtyp Tieren erhalten. Wie erwartet wurde IκBαΔN Protein in IκBαΔN Mäusen in allen untersuchten Geweben exprimiert. Embryonale Fibroblasten (MEF) wurden dann aus IκBαΔN Tieren isoliert und auf Induktion von NF-κB durch Zytokine untersucht (Abb. 1D). Die TNFα-induzierte NF-κB DNA Bindungsaktivität war stark unterdrückt, während IL-1β -induzierte Aktivität völlig inhibiert war (Abb. 1D). Das endogene IκBα Protein der MEF wurde nach IL-1β oder TNFα Stimulation degradiert, während, wie erwartet, das IκBαΔN Protein, dem Degradationssignale fehlen, stabil blieb (Abb. 1E).ES cell clones obtained by homologous recombination, which were IκBαΔN or loxPIκBαΔN positive according to Southern blot analyzes, were examined in Western blot analyzes for IκBα and IκBαΔN protein expression ( FIG. 1B). As expected, only IκBαΔN positive ES cells express the IκBαΔN protein. loxPIκBαΔN positive ES cells do not express IκBαΔN because there was no Cre-mediated recombination. The efficiency of IκBαΔN-mediated inhibition of NF-κB DNA binding activity was investigated in gel retardation assays ( Fig. 1C). While NF-κB p50-p65 binding activity in loxPIκBαΔN positive ES cells could be induced by PMA, no induced binding activity could be found in IκBαΔN positive ES cells ( Fig. 1C). IκBαΔN and loxPIκBαΔN ES cell clones were then used to generate chimeric mice by blastocyst injection. Heterozygous mice were obtained by subsequently crossing the chimeras with C57B1 / 6 wild type animals. As expected, IκBαΔN protein was expressed in IκBαΔN mice in all tissues examined. Embryonic fibroblasts (MEF) were then isolated from IκBαΔN animals and examined for the induction of NF-κB by cytokines ( Fig. 1D). The TNFα-induced NF-κB DNA binding activity was strongly suppressed, while IL-1β-induced activity was completely inhibited ( Fig. 1D). The endogenous IκBα protein of the MEF was degraded after IL-1β or TNFα stimulation, while, as expected, the IκBαΔN protein, which lacks degradation signals, remained stable ( Fig. 1E).
Das β-Catenin Gen wird ubiquitär und während der gesamten Embryonalentwicklung exprimiert. Da β-Catenin vorwiegend auf Ebene der Proteinstabilität reguliert wird (Hülsken et al. 2000), blieben die β-Catenin Proteinmengen in allen untersuchten Geweben und in MEF aus IκBαΔN-positiven Mäusen in gleicher Höhe wie in Wildtyp Tieren (Abb. 1E und nicht gezeigte Daten). Die Inaktivierung eines der beiden β-Catenin Allele hat also keinen Effekt auf die β-Catenin Proteinexpression. The β-catenin gene is expressed ubiquitously and throughout embryonic development. Since β-catenin is mainly regulated at the level of protein stability (Hülsken et al. 2000), the β-catenin protein levels in all examined tissues and in MEF from IκBαΔN-positive mice remained at the same level as in wild-type animals ( Fig. 1E and not shown data). The inactivation of one of the two β-catenin alleles has no effect on the β-catenin protein expression.
Diese molekularen Daten weisen also nach, daß IκBαΔN aus dem β-Catenin Lokus ubiquitär exprimiert wird und NF-κB effizient reprimiert und daß das loxPIκBαΔN Konstrukt ohne Cre Rekombination kein IκBαΔN exprimiert. Ferner wird β-Catenin Protein-Expression durch die mono-allelische Integration nicht beeinflußt.These molecular data thus demonstrate that IκBαΔN from the β-catenin locus is ubiquitously expressed and NF-κB efficiently repressed and that the loxPIκBαΔN construct no IκBαΔN expressed without Cre recombination. Furthermore, β-catenin protein expression not influenced by the mono-allelic integration.
Die IκBαΔN Mäuse weisen eine Reihe von pathologischen Merkmalen auf (Tabelle 1), von denen nur wenige von bisherigen Knockout Mäusen der verschiedenen NF-κB Untereinheiten bekannt sind (Gerondakis et al. 1999).The IκBαΔN mice have a number of pathological features (Table 1), of which only a few from previous knockout mice of the different NF-κB Subunits are known (Gerondakis et al. 1999).
Die loxPIκBαΔN Mäuse können jedoch phänotypisch nicht von Wildtyp-Mäusen unterschieden werden (Tabelle 1). Daher sind alle Phänotypen, die nach IκBαΔN Expression beobachtet werden, auf das IκBαΔN Protein und nicht auf Veränderungen des β-Catenin Lokus zurückzuführen. Wenn loxPIκBαΔN Mäuse jedoch mit deleter-Cre Mäusen (Schwenk et al., 1995), die das Cre Enzym ubiquitär in allen Körperzellen exprimieren, gepaart werden, zeigen die Nachkommen den gleichen Phänotyp wie IκBαΔN Mäuse (Tabelle 1). Das loxPIκBαΔN Konstrukt läßt sich also effizient durch Cre aktivieren. Hiermit ist sichergestellt, daß loxPIκBαΔN Mäuse sich für weitere Untersuchungen eignen, bei denen beliebige andere Cre-Mäuse verwendet werden, bei denen Cre in unterschiedlichster Art und Weise exprimiert wird.The loxPIκBαΔN mice, however, cannot phenotypically from wild-type mice can be distinguished (Table 1). Therefore, all phenotypes are based on IκBαΔN expression be observed on the IκBαΔN protein and not on changes in the β-catenin Locus traced. However, if loxPIκBαΔN mice with deleter-Cre mice (Schwenk et al., 1995), which ubiquitously express the Cre enzyme in all body cells, are paired, the offspring show the same phenotype as IκBαΔN mice (Table 1). The loxPIκBαΔN construct can thus be activated efficiently by Cre. This ensures that loxPIκBαΔN mice are suitable for further studies in which any other Cre mice are used in which Cre expresses in a variety of ways becomes.
Barnes, P. J. & Karin, M. Nuclear factor-kappaB: a pivotal transcription factor in chronic
inflammatory diseases (1997). N Engl J Med 336, 1066-71
DiSanto, J. P., Muller, W., Guy Grand, D., Fischer, A., and Rajewsky, K. (1995) Lymphoid
development in mice with a targeted deletion of the interleukin 2 receptor gamma chain.
Proc. Natl. Acad. Sci. USA 92, 377-381.
Gerondakis, S., Grossmann, M., Nakamura, Y., Pohl, T. & Grumont, R. (1999) Genetic
approaches in mice to understand Rel/NF-kappaB and IkappaB function: transgenics
and knockouts. Oncogene 18, 6888-95
Gu, H., Marth, J. D., Orban, P. C., Mossmann, H., and Rajewsky, K. (1994) Deletion of a DNA
polymerase beta gene segment in T cells using cell type-specific gene targeting.
Science 265, 103-106.
Haegel, H., Larue, L., Ohsugi, M., Fedorov, L., Herrenknecht, K., and Kemler, R. (1995)
Lack of beta-catenin affects mouse development at gastrulation. Development 121,
3529-3537.
Hatada, E. N., Krappmann, D., and Scheidereit, C. (2000) NF-κB and the innate immune
response. Curr. Opin. Immunol. 12, 52-8.
Hogan, B, Beddington, R. Costantini, F., Lacey, E. (1994) Manipulating the Mouse Embryo
Cold Spring Harbor Laboratory Press, New York,
Huelsken J, Vogel R, Brinkmann V, Erdmann B, Birchmeier C, Birchmeier W. (2000)
Requirement for beta-catenin in anterior-posterior axis formation in mice. J Cell Biol
148, 567-78
Hülsken, J., Birchmeier, W., and Behrens, J. (1994) E-cadherin and APC compete for the
interaction with beta-catenin and the cytoskeleton. J. Cell. Biol. 127, 2061-2069.
Krappmann, D., Wulczyn, F. G. and Scheidereit, C. (1996) Different mechanisms control
signal-induced degradation and basal turnover of the NF-κB inhibitor IκBα in vivo.
EMBD J. 15, 6716-6726.
Mayo, M. W. & Baldwin, A. S. (2000) The transcription factor NF-kappaB: control of
oncogenesis and cancer therapy resistance. Biochim Biophys Acta 1470, M55-62
Schwenk, F., Baron, U., and Rajewsky, K. (1995) A cre-transgenic mouse strain for the
ubiquitous deletion of loxP-flanked gene segments including deletion in germ cells. Nucl.
Acids Res. 23, 5080-5081.
Wang, C. Y., Cusack, J. C., Jr., Liu, R. & Baldwin, A. S., Jr. (1999) Control of inducible
chemoresistance: enhanced anti-tumor therapy through increased apoptosis by
inhibition of NF-kappaß. Nat Med 5, 412-7
Wulczyn, F. G., Krappmann, D., and Scheidereit, C. (1996) The NF-κB/Rel and IκB gene
families: mediators of immune response and inflammation J. Mol. Med. 74, 749-769.
Zhang, Y., Riesterer, C., Ayrall, A. M., Sablitzky, F., Littlewood, T. D., and Reth, M. (1996)
Inducible site-directed recombination in mouse embryonic stem cells. Nucleic Acids
Res. 24, 543-548. Barnes, PJ & Karin, M. Nuclear factor-kappaB: a pivotal transcription factor in chronic inflammatory diseases (1997). N Engl J Med 336, 1066-71
DiSanto, JP, Muller, W., Guy Grand, D., Fischer, A., and Rajewsky, K. (1995) Lymphoid development in mice with a targeted deletion of the interleukin 2 receptor gamma chain. Proc. Natl. Acad. Sci. USA 92, 377-381.
Gerondakis, S., Grossmann, M., Nakamura, Y., Pohl, T. & Grumont, R. (1999) Genetic approaches in mice to understand Rel / NF-kappaB and IkappaB function: transgenics and knockouts. Oncogene 18, 6888-95
Gu, H., Marth, JD, Orban, PC, Mossmann, H., and Rajewsky, K. (1994) Deletion of a DNA polymerase beta gene segment in T cells using cell type-specific gene targeting. Science 265, 103-106.
Haegel, H., Larue, L., Ohsugi, M., Fedorov, L., Herrenknecht, K., and Kemler, R. (1995) Lack of beta-catenin affects mouse development at gastrulation. Development 121, 3529-3537.
Hatada, EN, Krappmann, D., and Scheidereit, C. (2000) NF-κB and the innate immune response. Curr. Opin. Immunol. 12, 52-8.
Hogan, B, Beddington, R. Costantini, F., Lacey, E. (1994) Manipulating the Mouse Embryo Cold Spring Harbor Laboratory Press, New York,
Huelsken J, Vogel R, Brinkmann V, Erdmann B, Birchmeier C, Birchmeier W. (2000) Requirement for beta-catenin in anterior-posterior axis formation in mice. J Cell Biol 148, 567-78
Hülsken, J., Birchmeier, W., and Behrens, J. (1994) E-cadherin and APC compete for the interaction with beta-catenin and the cytoskeleton. J. Cell. Biol. 127, 2061-2069.
Krappmann, D., Wulczyn, FG and Scheidereit, C. (1996) Different mechanisms control signal-induced degradation and basal turnover of the NF-κB inhibitor IκBα in vivo. EMBD J. 15, 6716-6726.
Mayo, MW & Baldwin, AS (2000) The transcription factor NF-kappaB: control of oncogenesis and cancer therapy resistance. Biochim Biophys Acta 1470, M55-62
Schwenk, F., Baron, U., and Rajewsky, K. (1995) A cre-transgenic mouse strain for the ubiquitous deletion of loxP-flanked gene segments including deletion in germ cells. Nucl. Acids Res. 23, 5080-5081.
Wang, CY, Cusack, JC, Jr., Liu, R. & Baldwin, AS, Jr. (1999) Control of inducible chemoresistance: enhanced anti-tumor therapy through increased apoptosis by inhibition of NF-kappaß. Nat Med 5, 412-7
Wulczyn, FG, Krappmann, D., and Scheidereit, C. (1996) The NF-κB / Rel and IκB gene families: mediators of immune response and inflammation J. Mol. Med. 74, 749-769.
Zhang, Y., Riesterer, C., Ayrall, AM, Sablitzky, F., Littlewood, TD, and Reth, M. (1996) Inducible site-directed recombination in mouse embryonic stem cells. Nucleic Acids Res. 24, 543-548.
Genkarten der für die homologe Rekombination verwendeten Targeting Konstrukte, des Wildtyp β-Catenin Lokus und des IκBαΔN Leserahmens. Die Integration der kodierenden cDNA der transdominanten negativen Mutante von humanem IκBα, IκBαΔN, in den β-Catenin Lokus durch homologe Rekombination. (A) Der murine β-Catenin Lokus und Targeting Vektoren. Das β-Catenin Gen enthält 16 Exons. IκBαΔN und loxP-IκBαΔN wurden im korrekten Leserahmen mit dem endogenen Translations-Startcodon im zweiten Exon des β-Catenin Gens eingefügt. Die inserierten cDNA Konstrukte ersetzen die Exons 3 bis 6 des β-Catenin Gens und bewirken damit eine komplette Inaktivierung des β-Catenin Allels (Huelsken, 2000). In der loxP-IκBαΔN cDNA ist ein Stopcodon, welches am Anfang des Leserahmens eingefügt wurde, durch zwei loxP Sequenzen flankiert (schwarze Pfeile). (B) Expression von IκBαΔN und loxPIκBαΔN. Western Blot Analyse. Durch homologe Rekombination erhaltene loxPIκBαΔN (D5-C7) und IκBαΔN (D7-H9) ES Zell-Klone wurden für die Herstellung von Gesamtzellextrakten verwendet. Der im Western Blot verwendete Antikörper (αC-21, #sc371, Santa Cruz) ist gegen den C-Terminus von IκBα gerichtet. ns, unspezifisch. Molekularmassen-Standards sind angegeben. (C) Repression der Phorbolester-stimulierten DNA Bindungsaktivität von NF-κB durch IκBαΔN. Gel Retardienings-Assay von IκBαΔN ES Klonen (G2 and G3), sowie von loxPIκBαΔN ES Klonen (B7 and A3) vor Cre-Rekombinase-vermittelter Rekombination. Die ES-Zellklone wurden, wie angegeben, mit Phorbolester (PMA) behandelt oder unbehandelt belassen. Zur Inhibition spezifischer DNA-NF-κB Komplexe wurde der Bindungsreaktion anti-p65 Antikörper zugesetzt, wie angegeben. Der Gel Retardierungs-Assay wurde wie in Krappmann et al., 1996, beschrieben, durchgeführt. (D) Repression der Zytokin-stimulierten DNA Bindungsaktivität von NF-κB durch IκBαΔN. Embryonale Fibroblasten (MEFs) von Wildtyp (WT) and IκBαΔN Mäusen wurden mit IL1-β and TNFα für die angegebenen Zeiten stimuliert und Extrakte im Gelretardierungs-Assay analysiert. (E) Die gleichen Extrakte wie in E wurden in Western Blots IκBα and β-Catenin Proteinexpression untersucht, wie angegeben.Gene maps of the targeting used for homologous recombination Constructs of the wild-type β-catenin locus and the IκBαΔN reading frame. The integration of the coding cDNA of the transdominant negative mutant of human IκBα, IκBαΔN, in the β-catenin locus by homologous recombination. (A) The murine β-catenin locus and Targeting vectors. The β-catenin gene contains 16 exons. IκBαΔN and loxP-IκBαΔN were in the correct reading frame with the endogenous translation start codon in the second Exon of the β-catenin gene inserted. The inserted cDNA constructs replace exons 3 to 6 of the β-catenin gene and thus completely inactivate the β-catenin Allels (Huelsken, 2000). In the loxP-IκBαΔN cDNA is a stop codon, which is at the beginning of the reading frame was flanked by two loxP sequences (black arrows). (B) Expression of IκBαΔN and loxPIκBαΔN. Western blot analysis. By homologous Recombination obtained loxPIκBαΔN (D5-C7) and IκBαΔN (D7-H9) ES cell clones were used for the production of whole cell extracts. The one in the Western blot Antibody used (αC-21, # sc371, Santa Cruz) is against the C-terminus of IκBα directed. ns, unspecific. Molecular mass standards are given. (C) repression of the Phorbol ester-stimulated DNA binding activity of NF-κB by IκBαΔN. gel Retardation assay of IκBαΔN ES clones (G2 and G3), as well as loxPIκBαΔN ES Cloning (B7 and A3) before Cre recombinase mediated recombination. The ES cell clones were treated with phorbol ester (PMA) or left untreated as indicated. to Inhibition of specific DNA-NF-κB complexes became the binding reaction anti-p65 Antibodies added as indicated. The gel retardation assay was as in Krappmann et al., 1996. (D) Repression of the cytokine-stimulated DNA Binding activity of NF-κB by IκBαΔN. Wild-type embryonic fibroblasts (MEFs) (WT) and IκBαΔN mice were treated with IL1-β and TNFα for the times indicated stimulated and extracts analyzed in the gel retardation assay. (E) The same extracts as in E, IκBα and β-catenin protein expression were examined in Western blots, such as specified.
Pathophysiologische Konsequenzen der ubiquitären Expression von IκBΔN. Alle
phänotypischen Veränderungen der IκBΔN Tiere wurden auch bei den Nachkommen von
Kreuzungen der loxPIκBΔN Mäuse mit deleter-Cre Mäusen erhalten. loxPIκBΔN Mäuse
weisen dagegen keine phänotypischen Veränderungen auf und sind von Wildtyp Mäusen
nicht zu unterscheiden.
Pathophysiological consequences of the ubiquitous expression of IκBΔN. All phenotypic changes in the IκBΔN animals were also obtained in the progeny from crosses of the loxPIκBΔN mice with deleter-Cre mice. loxPIκBΔN mice, on the other hand, show no phenotypic changes and are indistinguishable from wild-type mice.
Claims (8)
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE2000141842 DE10041842A1 (en) | 2000-08-25 | 2000-08-25 | Genetically modified, non-human mammal that contains an additional inducible regulator gene |
| PCT/DE2001/003158 WO2002016573A2 (en) | 2000-08-25 | 2001-08-24 | Genetically modified, non-human mammal containing an additional inducible regulator gene |
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|---|---|---|---|
| DE2000141842 DE10041842A1 (en) | 2000-08-25 | 2000-08-25 | Genetically modified, non-human mammal that contains an additional inducible regulator gene |
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| DE10041842A1 true DE10041842A1 (en) | 2002-03-07 |
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| WO (1) | WO2002016573A2 (en) |
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| AU2003286729A1 (en) * | 2002-10-25 | 2004-05-13 | University Of Massachusetts | Modulation of cellular proliferation |
| CN104335970B (en) * | 2013-08-05 | 2016-08-24 | 应哲康 | A kind of method building people's psoriasis mouse model and application thereof |
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| EP0779361A3 (en) * | 1995-12-15 | 1999-11-10 | F. Hoffmann-La Roche Ag | Truncated form of inhibitory kappa B protein (1kB), recombinant production and uses thereof |
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2000
- 2000-08-25 DE DE2000141842 patent/DE10041842A1/en not_active Withdrawn
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| WO2002016573A3 (en) | 2002-06-20 |
| WO2002016573A2 (en) | 2002-02-28 |
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