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WO1999045767A1 - Non-human transgenic mammal, method for producing same and its use - Google Patents

Non-human transgenic mammal, method for producing same and its use Download PDF

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Publication number
WO1999045767A1
WO1999045767A1 PCT/DE1999/000747 DE9900747W WO9945767A1 WO 1999045767 A1 WO1999045767 A1 WO 1999045767A1 DE 9900747 W DE9900747 W DE 9900747W WO 9945767 A1 WO9945767 A1 WO 9945767A1
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WO
WIPO (PCT)
Prior art keywords
human mammal
inducing function
function
mouse
cells
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
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PCT/DE1999/000747
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German (de)
French (fr)
Inventor
Holger Reichardt
Günther Schütz
Klaus KÄSTNER
Peter Herrlich
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Deutsches Krebsforschungszentrum DKFZ
Karlsruher Institut fuer Technologie KIT
Original Assignee
Deutsches Krebsforschungszentrum DKFZ
Forschungszentrum Karlsruhe GmbH
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Publication date
Application filed by Deutsches Krebsforschungszentrum DKFZ, Forschungszentrum Karlsruhe GmbH filed Critical Deutsches Krebsforschungszentrum DKFZ
Priority to JP2000535196A priority Critical patent/JP2002505858A/en
Priority to AU39249/99A priority patent/AU3924999A/en
Priority to EP99922034A priority patent/EP1061798A1/en
Publication of WO1999045767A1 publication Critical patent/WO1999045767A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • 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
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/721—Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
    • 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
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT

Definitions

  • the present invention relates to a non-human mammal that has an altered glucocorticoid receptor.
  • the invention further relates to a method for producing such a mammal and its use for testing chemicals, drugs and therapeutic approaches.
  • GR The glucocorticoid receptor
  • AP1 such as collagenase gene, or NFKB, such as TNF, II-2 or II-6.
  • GR GR-like repressing function
  • diseases are e.g. Disorders of the acute phase reaction, septic shock, asthma, acute shortness of breath syndrome, inflammatory diseases, autoimmune diseases, such as rheumatoid arthritis or multiple sclerosis, neuropsychiatric diseases, diseases of the hypothalamic-pituitary-adrenal axis, osteoporosis, diabetes, steroid -Myopathy or skin disorders.
  • diseases are e.g. Disorders of the acute phase reaction, septic shock, asthma, acute shortness of breath syndrome, inflammatory diseases, autoimmune diseases, such as rheumatoid arthritis or multiple sclerosis, neuropsychiatric diseases, diseases of the hypothalamic-pituitary-adrenal axis, osteoporosis, diabetes, steroid -Myopathy or skin disorders.
  • the present invention is therefore based on the object of providing a means with which the repressing function of GR can be selectively examined and, if necessary, interfered with.
  • the present invention thus relates to a non-human mammal, the GR of which is changed in its inducing function. In particular, the inducing function is switched off.
  • the present invention is based on the knowledge of the applicant that the individual functions of GR can be changed separately from one another. He found that the inductive function of GR can be changed, in particular switched off, without affecting the repressing function. He also recognized that the inducing function of GR is related to its dimerization or DNA binding and by mutation of this, e.g. B. in the D-Loop of GR, in particular by the point mutation A 458 T, the inducing function of GR changed, in particular can be switched off. Furthermore, he recognized that a GR that changed in its inducing function still has immunosuppressive and anti-inflammatory activity. The applicant has obtained his knowledge from a transgenic mouse, the GR of which has been changed in its inducing function (cf. Examples 2 and 3).
  • the knowledge of the applicant is used to provide a non-human mammal whose GR changes in its inducing function, in particular this is switched off.
  • non-human mammal includes any mammal whose GR may be altered in its inducing function. Examples of such mammals are mouse, rat, rabbit, horse, cattle, sheep, goat, monkey, pig, dog and cat, with mouse being preferred.
  • GR whose inducing function is changed, in particular switched off
  • the expression "GR, whose inducing function is changed, in particular switched off” includes a GR of a non-human mammal, the repressing function of which is unchanged, but which has a change in the inducing- - 3 - has the function.
  • the inductive function is preferably switched off. This can be achieved, for example, by mutating the dimerization of GR or its DNA binding. This is achieved, for example, by a mutation in the D loop of GR, in particular by the point mutation A 458 T.
  • Another object of the present invention are cells obtained from the above non-human mammal. These cells can be in any form, e.g. in a primary or long-term culture.
  • a non-human mammal according to the invention can be provided by conventional methods.
  • a method is favorable which comprises the following steps:
  • embryonic stem cells refers to any embryonic stem cells of a non-human mammal that are suitable for mutating the DNA coding for the inducing function of GR.
  • the embryonic stem cells are preferably from the mouse, in particular the cells E14 / 1.
  • vector includes any vector that is created by recombination with - 4 - allows the DNA of embryonic stem cells to change the DNA coding for the inductive function of GR.
  • the change is preferably such that the inductive function of GR is switched off.
  • the vector has a DNA which carries a mutation in an area which is necessary for the dimerization of GR or its DNA binding.
  • the mutation can lie in a region that codes for the D-loop of GR.
  • the mutation can be the point mutation A 458 T.
  • the vector has a marker with which it is possible to select for existing stem cells in which the desired recombination has taken place.
  • Such a marker is, for example, the loxP / tkneo cassette, which can be removed from the genome again using the Cre / loxP system.
  • a foregoing vector is also the subject of the present invention.
  • Such a vector ie the vector from example 1 or FIG. 1, was deposited with the DSMZ as pmGR2-tr under DSM 1 2026 on February 9, 998.
  • the present invention provides a non-human mammal, the GR of which is altered in its inducing function. This change can be a switching off of the inductive function. With such a mammal or cells from it, the repressing function of GR can be investigated selectively. It also makes it possible to find substances, drugs and therapeutic approaches that can be used to selectively influence the repressing function of GR.
  • the present invention therefore provides a basis for acting on a wide variety of diseases.
  • Such diseases include disorders of the acute phase reaction, septic shock, asthma, - 5 - Acute shortness of breath syndrome, inflammatory diseases, autoimmune diseases such as rheumatoid arthritis or multiple sclerosis, neuropsychiatric diseases, diseases of the hypothalamic-pituitary-adrenal axis, osteoporosis, diabetes, steroid myopathy or skin diseases.
  • Fig. 1 shows the introduction of the point mutation A 485 T in GR.
  • amino acid sequence of the second zinc finger in the DNA binding domain of GR is given.
  • the exchange of alanine 458 to threonine is indicated.
  • the strategy for recombination is given in (b).
  • the stars mark the position of the point mutation.
  • the modified locus represents the structure of the GR locus after recombination, the end locus the structure after the additional recombination of the two loxP sites (triangles) by Cre recombinase.
  • genotype is indicated by PCR.
  • a 240 base pair fragment is amplified using the primers indicated by arrows in (b) and cleaved by the restriction enzyme BsrG 1, for which a new recognition site has been introduced by the point mutation.
  • the two 120 bp fragments created by the point mutation are distinguished from the uncut fragment by gel electrophoresis.
  • a sequence comparison is given in (d). Brain RNA is amplified by RT-PCR using primers whose sequences are in exons 3 and 5 of GR. The fragments obtained are subcloned and sequenced. The two mutated bases are indicated.
  • FIG. 2 shows the comparison between a mouse according to the invention and a wild-type mouse with regard to the inducing function of GR.
  • TAT tyrosine aminotransferase
  • Dexamethasone is used in a mouse according to the invention - 6 - and injected into a wild-type mouse and liver mRNA expression of TAT or ⁇ -actin as a control is analyzed by Northern blot.
  • B indicates the induction of the proliferation of erythrocyte progenitor cells. Erythrocyte progenitor cells are isolated and cultured. Cumulative line numbers are determined. The induction of apoptosis of thymocytes is given in (c). The percentage of viable cells after treatment with dexamethasone (hatched bars) or in controls (filled bars) are given.
  • Figure 3 shows the induced inhibition of AP-1 induced repression of the collagenase gene.
  • Primary fibroblasts from a mouse according to the invention or a wild-type mouse are treated with dexamethasone (Dex), TPA, or both, and mRNA amounts of MMP-1 3 (mouse collagenase-3) and GAPDH as a control are monitored by Northern blot analyzed.
  • Dex dexamethasone
  • TPA TPA
  • GAPDH GAPDH
  • Figure 4 shows the induced expression of cytokine mRNA and its repression.
  • Primary thymocytes from a mouse according to the invention or a wild-type mouse are treated with ionomycin, phorbol ester (TPA), dexamethasone (dex) or a combination thereof (T + D).
  • TPA phorbol ester
  • dex dexamethasone
  • T + D The RNase protection assay is used to determine the mRNA expression of II-2 and TNF.
  • A), (C) relate to the induced expression and repression of II-2 mRNA and their quantification.
  • B), (D) relate to the induced expression and repression of IFN mRNA and their quantification,
  • con means control and (n, d) undetectable.
  • Figure 5 shows the expression of cytokine mRNA.
  • Macrophages from a mouse according to the invention or a wild-type mouse are treated with lipopolysaccharide (LPS) or a combination of LPS and dex (L + D).
  • LPS lipopolysaccharide
  • L + D dex
  • RNase-protection assay the - 7 - mRNA expression of TNF ⁇ and 11-6 determined.
  • A), (B), (C) relate to the expression and repression of 11-6 or TNF ⁇ mRNA and their quantification, (con) means control.
  • Fig. 6 shows the response of a mouse to infection or inflammation.
  • a mouse according to the invention or a wild-type mouse is treated with LPS and the serum concentration of TNF 0 is determined.
  • B) The skin of a mouse according to the invention or a wild-type mouse is treated with TPA or a combination of TPA and dexamethasone (T + D) and the serum concentration of 11-6 is determined.
  • Example 1 Provision of a non-human mammal according to the invention
  • a non-human mammal whose GR is switched off in its inducing function.
  • a vector which has approximately 1 1 kb of the GR gene, including exons 3 and 4.
  • the vector in exon 4 has a point mutation, as a result of which the GR encoded thereby at position 458 has threonine instead of alanine.
  • the vector comprises a loxP-tkneo selection cassette (see FIG. 1).
  • This vector is introduced into the mouse embryonic stem cells E14 / 1 by electroporation.
  • Stably transfected cells are obtained by selection with 350 ⁇ g / ml G41 8. These cells are transiently transfected with 20 ⁇ g of a Cre expression plasmid before being given 1 / M Gancyclovir - 8 - is added. This selects for cells that have lost the selection cassette. These cells have the specified point mutation in the GR gene and additionally about 50 base pairs along the remaining lox P site in intron 3.
  • mice are injected into mouse blastocysts and then introduced into female mice. Chimeric mice are obtained, from which heterozygous and homozygous mice are obtained by mating.
  • Example 2 Detection of individual functions of a non-human mammal according to the invention
  • Example 1 The non-human mammal provided in Example 1 is used. This is used in test trials that are specific to the individual functions of GR.
  • a mouse according to the invention and a wild-type mouse are each injected with 10 yg / 100 g dexamethasone or PBS.
  • the animals are killed after two hours.
  • Liver RNA is isolated and the mRNA expression of tyrosine amino transferase (TAT) or of ⁇ -actin as a control is determined in a Northern blot (cf. FIG. 2a).
  • TAT tyrosine amino transferase
  • ⁇ -actin as a control is determined in a Northern blot (cf. FIG. 2a).
  • fetal liver is isolated from a mouse and a wild-type mouse embryo according to the invention and dispersed in individual cells. These are then in - 9 - Modified CFU-E medium containing SCF, hEpo, IGF-1 and dexamethasone increased. A growth kinetics is created by counting the cells daily using an electronic cell counter (see FIG. 2b).
  • a wild-type mouse but not a mouse according to the invention, can induce the proliferation of erythrocyte precursor cells.
  • Thymocytes are isolated from a 6-1 2 week old mouse according to the invention and a correspondingly old wild-type mouse and cultured in RPMI medium for 24 hours in the presence or absence of dexamethasone (10 "6 M). The cells are then stained with propidium iodide and analyzed for their fluorescence intensity using FACS. The loss of DNA content is taken as a measure of apoptosis.
  • a wild-type mouse but not a mouse according to the invention, can induce the apoptosis of thymocytes.
  • Primary fibroblasts are isolated from embryos of a mouse according to the invention and a wild-type mouse. The fibroblasts are treated for 6 hours with 10 "6 M dexamethasone, 10 " 7 M TPA or both before RNA is isolated and the mRNA expression of MMP-1 3 (mouse collagenase 3 gene) and GAPDH as a control in a Northern -Blot can be determined (see FIG. 3).
  • a wild-type mouse and also a mouse according to the invention can inhibit the AP1-related activation of the collagenase gene.
  • a mammal according to the invention for example a mouse, - 10 - expresses a GR in which the repressing function is retained, but the inducing function is changed, in particular switched off.
  • Example 3 Investigations on a non-human mammal according to the invention or cells thereof with regard to immunosuppressive or anti-inflammatory activity
  • thymocytes from a mouse according to the invention or a wild-type mouse are isolated and 6 h with 0.5 ⁇ g / ml ionomycin and 10 ⁇ g / ml phorbol ester (TPA) or 10 ⁇ 6 M dexamethasone (dex) or a combination thereof (T + D) treated. 2 ⁇ g total RNA are each subjected to an “RNase protection assay”.
  • Peritoneal macrophages from a mouse according to the invention or a wild-type mouse are isolated 5 d after a thioglycolate treatment and after 24 h for 2 h with 100 ng / ml lipopolysaccharide (LPS) or a combination of 10 "6 M dexamethasone and lipopolysaccharide ( L + D) 0.5 ⁇ g of total RNA is subjected to an "RNAse protection assay".
  • LPS lipopolysaccharide
  • L + D lipopolysaccharide
  • a mouse according to the invention or a wild-type mouse are administered in each case 10 ⁇ g LPS.
  • the mice are sacrificed at 0, 60 min and 1 80 min.
  • Serum is isolated in each case and the TNF ⁇ concentration is determined using a conventional ELISA kit.
  • the left ear of a mouse according to the invention or a wild-type mouse is in each case treated with TPA or a combination of TPA and dexamethasone (T + D). A certain area of the ear is removed and the weight of the edema formed is determined.
  • mice according to the invention and its cells have immunosuppressive and anti-inflammatory activity. This is comparable to that of a wild-type mouse.

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Abstract

The invention relates to a non-human mammal, whose glucocorticoid receptor (GR) has been modified in its inducing function. The invention also relates to a method for producing such a mammal and to its use for testing chemicals, medicines and new therapies.

Description

NICHT-MENSCHLICHES TRASGENISCHES SÄUGETIER , SOWIE VERFAHREN ZU SEINER HERSTELLUNG UND VERWENDUNG NON-HUMAN TRASGENIC MAMMAL, AND METHOD FOR THE PRODUCTION AND USE THEREOF

Die vorliegende Erfindung betrifft ein nicht-menschliches Säugetier, das einen veränderten Glukokortikoid-Rezeptor aufweist. Ferner betrifft die Erfindung ein Verfahren zur Herstellung eines solchen Säugetiers sowie dessen Verwendung zur Testung von Chemikalien, Arzneimitteln und Therapieansätzen.The present invention relates to a non-human mammal that has an altered glucocorticoid receptor. The invention further relates to a method for producing such a mammal and its use for testing chemicals, drugs and therapeutic approaches.

Der Glukokortikoid-Rezeptor (nachstehend mit GR bezeichnet) ist ein in vielen Zellen vorliegender Rezeptor, der durch Bindung von Glukokortikoiden aktiviert wird. In dieser Form induziert GR die Expression von Zielgenen. Solche sind z.B. Gene, deren Produkte in der Glukoneogenese, der Proliferation von Erythrozyten- Vorläuferzellen oder der Apoptose von Thymozyten eine Rolle spielen. Andererseits reprimiert aktivierter GR auch die Expression von Zielgenen. Solche sind z.B. Gene, die von AP1 , wie Kollagenase-Gen, oder NFKB, wie TNF , II-2 oder II- 6, aktiviert werden.The glucocorticoid receptor (hereinafter referred to as GR) is a receptor that is present in many cells and is activated by the binding of glucocorticoids. In this form, GR induces the expression of target genes. Such are e.g. Genes whose products play a role in gluconeogenesis, the proliferation of erythrocyte progenitor cells or the apoptosis of thymocytes. On the other hand, activated GR also represses the expression of target genes. Such are e.g. Genes that are activated by AP1, such as collagenase gene, or NFKB, such as TNF, II-2 or II-6.

Es wird angedacht, selektiv in die Funktionen von GR einzugreifen. Insbesondere scheint die reprimierende Funktion hierfür interessant zu sein, da sie eine mögliche Ansatzstelle für das Eingreifen in die verschiedensten Erkrankungen gibt. Solche Erkrankungen sind z.B. Störungen der Akut-Phase-Reaktion, septischer Schock, Asthma, akutes Atemnot-Syndrom, inflammatorische Erkrankungen, Autoimmunerkrankungen, wie rheumatoide Arthritis oder multiple Sklerose, neuropsychiatrische Erkrankungen, Erkrankungen der Hypothalamus-Hypophy- sen-Nebennieren-Achse, Osteoporose, Diabetes, Steroid-Myopathie oder Hauterkrankungen.It is planned to intervene selectively in the functions of GR. In particular, the repressing function seems to be interesting because it provides a possible starting point for intervening in a wide variety of diseases. Such diseases are e.g. Disorders of the acute phase reaction, septic shock, asthma, acute shortness of breath syndrome, inflammatory diseases, autoimmune diseases, such as rheumatoid arthritis or multiple sclerosis, neuropsychiatric diseases, diseases of the hypothalamic-pituitary-adrenal axis, osteoporosis, diabetes, steroid -Myopathy or skin disorders.

Der vorliegenden Erfindung liegt daher die Aufgabe zugrunde, ein Mittel bereitzustellen, mit dem selektiv die reprimierende Funktion von GR untersucht und gegebenenfalls in sie eingegriffen werden kann.The present invention is therefore based on the object of providing a means with which the repressing function of GR can be selectively examined and, if necessary, interfered with.

Erfindungsgemäß wird dies durch die Gegenstände in den Patentansprüchen - 2 - erreicht.According to the invention, this is the subject of the claims - 2 - reached.

Gegenstand der vorliegenden Erfindung ist somit ein nicht-menschliches Säugetier, dessen GR in seiner induzierenden Funktion verändert ist. Insbesondere ist die induzierende Funktion ausgeschaltet.The present invention thus relates to a non-human mammal, the GR of which is changed in its inducing function. In particular, the inducing function is switched off.

Die vorliegende Erfindung beruht auf der Erkenntnis des Anmelders, daß die einzelnen Funktionen von GR getrennt voneinander verändert werden können. Er hat gefunden, daß die induzierende Funktion von GR verändert, insbesondere ausgeschaltet werden kann, ohne daß hiervon die reprimierende Funktion betroffen wird. Ferner hat er erkannt, daß die induzierende Funktion von GR mit seiner Dimerisierung bzw. DNA-Bindung zusammenhängt und durch Mutation dieser, z. B. im D-Loop von GR, insbesondere durch die Punktmutation A 458 T, die induzierende Funktion von GR verändert, insbesondere ausgeschaltet werden kann. Desweiteren hat er erkannt, daß ein in seiner induzierenden Funktion veränderter GR nachwievor immunsuppressive und entzündungshemmende Aktivität aufweist. Der Anmelder hat seine Erkenntnisse durch eine transgene Maus erhalten, deren GR in seiner induzierenden Funktion verändert ist (vgl. Beispiele 2 und 3) .The present invention is based on the knowledge of the applicant that the individual functions of GR can be changed separately from one another. He found that the inductive function of GR can be changed, in particular switched off, without affecting the repressing function. He also recognized that the inducing function of GR is related to its dimerization or DNA binding and by mutation of this, e.g. B. in the D-Loop of GR, in particular by the point mutation A 458 T, the inducing function of GR changed, in particular can be switched off. Furthermore, he recognized that a GR that changed in its inducing function still has immunosuppressive and anti-inflammatory activity. The applicant has obtained his knowledge from a transgenic mouse, the GR of which has been changed in its inducing function (cf. Examples 2 and 3).

Erfindungsgemäß werden die Erkenntnisse des Anmelders genutzt, ein nichtmenschliches Säugetier bereitzustellen, dessen GR in seiner induzierenden Funktion verändert, insbesondere diese ausgeschaltet ist.According to the invention, the knowledge of the applicant is used to provide a non-human mammal whose GR changes in its inducing function, in particular this is switched off.

Der Ausdruck "nicht-menschliches Säugetier" umfaßt jegliches Säugetier, dessen GR in seiner induzierenden Funktion verändert sein kann. Beispiele solcher Säugetiere sind Maus, Ratte, Kaninchen, Pferd, Rind, Schaf, Ziege, Affe, Schwein, Hund und Katze, wobei Maus bevorzugt ist.The term "non-human mammal" includes any mammal whose GR may be altered in its inducing function. Examples of such mammals are mouse, rat, rabbit, horse, cattle, sheep, goat, monkey, pig, dog and cat, with mouse being preferred.

Der Ausdruck "GR, dessen induzierende Funktion verändert, insbesondere ausgeschaltet ist" umfaßt einen GR eines nicht-menschliches Säugetiers, dessen reprimierende Funktion unverändert ist, das aber eine Veränderung in der induzieren- - 3 - den Funktion aufweist. Vorzugsweise ist die induzierende Funktion ausgeschaltet. Dies kann z.B. dadurch erreicht werden, daß die Dimerisierung von GR bzw. seine DNA-Bindung mutiert wird. Solches wird z.B. durch eine Mutation im D- Loop von GR, insbesondere durch die Punktmutation A 458 T erzielt.The expression "GR, whose inducing function is changed, in particular switched off" includes a GR of a non-human mammal, the repressing function of which is unchanged, but which has a change in the inducing- - 3 - has the function. The inductive function is preferably switched off. This can be achieved, for example, by mutating the dimerization of GR or its DNA binding. This is achieved, for example, by a mutation in the D loop of GR, in particular by the point mutation A 458 T.

Ein weiterer Gegenstand der vorliegenden Erfindung sind Zellen, die aus dem vorstehenden nicht-menschlichen Säugetier erhalten werden. Diese Zellen können in jeglicher Form vorliegen, z.B. in einer Primär- oder Langzeit-Kultur.Another object of the present invention are cells obtained from the above non-human mammal. These cells can be in any form, e.g. in a primary or long-term culture.

Ein erfindungsgemäßes nicht-menschliches Säugetier kann durch übliche Verfahren bereitgestellt werden. Günstig ist ein Verfahren, das folgende Schritte umfaßt:A non-human mammal according to the invention can be provided by conventional methods. A method is favorable which comprises the following steps:

(a) Transfektion von embryonalen Stammzellen eines nicht-menschlichen Säugetiers mit einem Vektor, der eine Rekombination zwischen der für die induzierende Funktion von GR kodierenden DNA der embryonalen Stammzellen und einer entsprechenden mutierten DNA des Vektors ermöglicht,(a) transfection of embryonic stem cells of a non-human mammal with a vector which enables a recombination between the DNA of the embryonic stem cells coding for the inducing function of GR and a corresponding mutated DNA of the vector,

(b) Isolierung von in (a) stabil transfizierten Zellen und Einbringung dieser in weibliche Tiere eines nicht-menschlichen Säugetiers, sowie(b) isolation of cells stably transfected in (a) and introduction into female animals of a non-human mammal, and

(c) Analyse der in (b) erhaltenen Nachkommen auf einen GR, dessen induzierende Funktion verändert, insbesondere ausgeschaltet ist.(c) Analysis of the offspring obtained in (b) for a GR whose inductive function changes, in particular is switched off.

Für die Ausdrücke "nicht-menschliches Säugetier" und "GR, dessen induzierende Funktion verändert, insbesondere ausgeschaltet ist" gelten die vorstehenden Ausführungen entsprechend.The above statements apply accordingly to the terms “non-human mammal” and “GR, the inductive function of which changes, in particular is switched off”.

Ferner betrifft der Ausdruck "embryonale Stammzellen" jegliche embryonalen Stammzellen eines nicht-menschlichen Säugetiers, die sich zur Mutierung der für die induzierende Funktion von GR kodierenden DNA eignen. Vorzugsweise sind die embryonalen Stammzellen von der Maus, insbesondere die Zellen E14/1 .Furthermore, the term "embryonic stem cells" refers to any embryonic stem cells of a non-human mammal that are suitable for mutating the DNA coding for the inducing function of GR. The embryonic stem cells are preferably from the mouse, in particular the cells E14 / 1.

Der Ausdruck "Vektor" umfaßt jeglichen Vektor, der durch Rekombination mit - 4 - der DNA von embryonalen Stammzellen eine Veränderung der für die induzierende Funktion von GR kodierenden DNA ermöglicht. Vorzugsweise ist die Veränderung dergestalt, daß die induzierende Funktion von GR ausgeschaltet ist. Günstig ist es, wenn der Vektor eine DNA aufweist, die eine Mutation in einem Bereich trägt, der für die Dimerisierung von GR bzw. seine DNA-Bindung notwendig ist. Insbesondere kann die Mutation in einem Bereich liegen, der für den D-Loop von GR kodiert. Ganz besonders kann die Mutation die Punktmutation A 458 T sein. Ferner ist es günstig, wenn der Vektor einen Marker aufweist, mit dem auf vorhandene Stammzellen selektioniert werden kann, in denen die gewünschte Rekombination erfolgt ist. Ein solcher Marker ist z.B. die loxP/tkneo- Cassette, die mit Hilfe des Cre/loxP-Systems wieder aus dem Genom entfernt werden kann. Ein vorstehender Vektor ist ebenfalls Gegenstand der vorliegenden Erfindung. Ein solcher Vektor, d.h der Vektor von Beispiel 1 bzw. Fig. 1 wurde, bei der DSMZ als pmGR2-tr unter DSM 1 2026 am 1 9. Februar 1 998 hinterlegt.The term "vector" includes any vector that is created by recombination with - 4 - allows the DNA of embryonic stem cells to change the DNA coding for the inductive function of GR. The change is preferably such that the inductive function of GR is switched off. It is favorable if the vector has a DNA which carries a mutation in an area which is necessary for the dimerization of GR or its DNA binding. In particular, the mutation can lie in a region that codes for the D-loop of GR. In particular, the mutation can be the point mutation A 458 T. Furthermore, it is favorable if the vector has a marker with which it is possible to select for existing stem cells in which the desired recombination has taken place. Such a marker is, for example, the loxP / tkneo cassette, which can be removed from the genome again using the Cre / loxP system. A foregoing vector is also the subject of the present invention. Such a vector, ie the vector from example 1 or FIG. 1, was deposited with the DSMZ as pmGR2-tr under DSM 1 2026 on February 9, 998.

Desweiteren kennt der Fachmann Bedingungen und Materialien, um die Schritte (a)-(c) durchzuführen. Hinsichtlich der Analyse in (c) wird er z.B. an Verfahren denken, mit denen er nachweisen kann, daß die Induzierung von Genen abgeschwächt bzw. verhindert ist, deren Produkte in der Glukoneogenese, der Proliferation von Erythrozyten-Vorläuferzellen oder der Apoptose von Thymozy- ten eine Rolle spielen. Solche Verfahren werden nachstehend in den Beispielen beschrieben.Furthermore, the person skilled in the art knows the conditions and materials in order to carry out steps (a) - (c). Regarding the analysis in (c) it will e.g. think of methods by which he can demonstrate that the induction of genes is weakened or prevented, the products of which play a role in gluconeogenesis, the proliferation of erythrocyte progenitor cells or the apoptosis of thymocytes. Such methods are described below in the examples.

Mit der vorliegenden Erfindung wird ein nicht-menschliches Säugetier bereitgestellt, dessen GR in seiner induzierenden Funktion verändert ist. Diese Veränderung kann ein Ausschalten der induzierenden Funktion sein. Mit einem solchen Säugetier bzw. Zellen daraus kann selektiv die reprimierende Funktion von GR untersucht werden. Ferner ist es hiermit möglich, Substanzen, Arzneimittel und Therapieansätze zu finden, mit denen selektiv auf die reprimierende Funktion von GR eingewirkt werden kann. Daher liefert die vorliegende Erfindung eine Basis, um auf die verschiedensten Erkrankungen einzuwirken. Solche Erkrankungen sind z.B. Störungen der Akut-Phase-Reaktion, septischer Schock, Asthma, - 5 - akutes Atemnot-Syndrom, inflammatorische Erkrankungen, Autoimmunerkrankungen, wie rheumatoide Arthritis oder multiple Sklerose, neuropsychiatrische Erkrankungen, Erkrankungen der Hypothalamus-Hypophysen-Nebennieren-Achse, Osteoporose, Diabetes, Steroid-Myopathie oder Hauterkrankungen.The present invention provides a non-human mammal, the GR of which is altered in its inducing function. This change can be a switching off of the inductive function. With such a mammal or cells from it, the repressing function of GR can be investigated selectively. It also makes it possible to find substances, drugs and therapeutic approaches that can be used to selectively influence the repressing function of GR. The present invention therefore provides a basis for acting on a wide variety of diseases. Such diseases include disorders of the acute phase reaction, septic shock, asthma, - 5 - Acute shortness of breath syndrome, inflammatory diseases, autoimmune diseases such as rheumatoid arthritis or multiple sclerosis, neuropsychiatric diseases, diseases of the hypothalamic-pituitary-adrenal axis, osteoporosis, diabetes, steroid myopathy or skin diseases.

Kurze Beschreibung der Zeichnungen:Brief description of the drawings:

Fig. 1 zeigt die Einführung der Punktmutation A 485 T in GR. In (a) wird die Aminosäuresequenz des zweiten Zinkfingers in der DNA-Bindungsdomäne von GR angegeben. Der Austausch von Alanin 458 zu Threonin ist angegeben. In (b) wird die Strategie für die Rekombination angegeben. Die Sterne markieren die Position der Punktmutation. Der modifizierte Locus repräsentiert die Struktur des GR- Locus nach Rekombination, der End-Locus die Struktur nach der zusätzlichen Rekombination der zwei loxP-Stellen (Dreiecke) durch Cre-Rekombinase. In (c) wird der Genotyp durch PCR angegeben. Ein 240 Basenpaar-Fragment wird unter der Verwendung der in (b) durch Pfeile angegebenen Primer amplifiziert und durch das Restriktionsenzym BsrG 1 gespalten, für das eine neue Erkennungsstelle durch die Punktmutation eingeführt worden ist. Die zwei 1 20 bp- Fragmente, die durch die Punktmutation geschaffen worden sind, werden von dem ungeschnittenen Fragment durch Gelelektrophorese unterschieden. In (d) wird ein Sequenzvergleich angegeben. Gehirn-RNA wird durch RT-PCR amplifiziert, wobei Primer verwendet werden, deren Sequenzen in den Exons 3 und 5 von GR vorliegen. Die erhaltenen Fragmente werden subkloniert und sequenziert. Die zwei mutierten Basen sind angegeben.Fig. 1 shows the introduction of the point mutation A 485 T in GR. In (a) the amino acid sequence of the second zinc finger in the DNA binding domain of GR is given. The exchange of alanine 458 to threonine is indicated. The strategy for recombination is given in (b). The stars mark the position of the point mutation. The modified locus represents the structure of the GR locus after recombination, the end locus the structure after the additional recombination of the two loxP sites (triangles) by Cre recombinase. In (c) the genotype is indicated by PCR. A 240 base pair fragment is amplified using the primers indicated by arrows in (b) and cleaved by the restriction enzyme BsrG 1, for which a new recognition site has been introduced by the point mutation. The two 120 bp fragments created by the point mutation are distinguished from the uncut fragment by gel electrophoresis. A sequence comparison is given in (d). Brain RNA is amplified by RT-PCR using primers whose sequences are in exons 3 and 5 of GR. The fragments obtained are subcloned and sequenced. The two mutated bases are indicated.

Fig. 2 zeigt den Vergleich zwischen einer erfindungsgemäßen Maus und einer Wildtyp-Maus hinsichtlich der induzierenden Funktion von GR. In (a) wird die Induzierung von Tyrosinaminotransferase (TAT) angegeben. Dexamethason wird in eine erfindungsgemäße Maus - 6 - und in eine Wildtyp-Maus injiziert und Leber-mRNA-Expression von TAT bzw. ß-Aktin als Kontrolle wird durch Northern Blot analysiert. In (b) wird die Induzierung der Proliferation von Erythrozyten-Vorläuferzellen angegeben. Erythrozyten-Vorläuferzellen werden isoliert und kultiviert. Kumulative Zeil-Zahlen werden bestimmt. In (c) wird die Induzierung der Apoptose von Thymozyten angegeben. Der Prozentsatz von lebensfähigen Zellen nach Behandlung mit Dexa- methason (schraffierte Balken) oder in Kontrollen (ausgefüllte Balken) sind angegeben.2 shows the comparison between a mouse according to the invention and a wild-type mouse with regard to the inducing function of GR. In (a) the induction of tyrosine aminotransferase (TAT) is given. Dexamethasone is used in a mouse according to the invention - 6 - and injected into a wild-type mouse and liver mRNA expression of TAT or β-actin as a control is analyzed by Northern blot. (B) indicates the induction of the proliferation of erythrocyte progenitor cells. Erythrocyte progenitor cells are isolated and cultured. Cumulative line numbers are determined. The induction of apoptosis of thymocytes is given in (c). The percentage of viable cells after treatment with dexamethasone (hatched bars) or in controls (filled bars) are given.

Fig. 3 zeigt die induzierte Inhibierung der AP-1 bedingten Reprimierung des Kollagenase-Gens. Primäre Fibroblasten aus einer erfindungsgemäßen Maus bzw. einer Wildtyp-Maus werden mit Dexametha- son (Dex), TPA, oder beidem behandelt und mRNA-Mengen von MMP-1 3 (Maus-Kollagenase-3) und GAPDH als Kontrolle werden durch Northern Blot analysiert.Figure 3 shows the induced inhibition of AP-1 induced repression of the collagenase gene. Primary fibroblasts from a mouse according to the invention or a wild-type mouse are treated with dexamethasone (Dex), TPA, or both, and mRNA amounts of MMP-1 3 (mouse collagenase-3) and GAPDH as a control are monitored by Northern blot analyzed.

Fig. 4 zeigt die induzierte Expression von Cytokin-mRNA und ihre Reprimierung. Primäre Thymozyten aus einer erfindungsgemäßen Maus bzw. einer Wildtyp-Maus werden mit lonomycin, Phorbolester (TPA), Dexamethason (dex) bzw. einer Kombination daraus (T + D) behandelt. Mittels "RNase protection assay" wird die mRNA-Ex- pression von II-2 und TNF bestimmt. (A), (C) betreffen die induzierte Expression und Reprimierung von II-2 mRNA und ihre Quantifizierung. (B), (D) betreffen die induzierte Expression und Reprimierung von IFN mRNA und ihre Quantifizierung, (con) bedeutet Kontrolle und (n, d) nicht nachweisbar.Figure 4 shows the induced expression of cytokine mRNA and its repression. Primary thymocytes from a mouse according to the invention or a wild-type mouse are treated with ionomycin, phorbol ester (TPA), dexamethasone (dex) or a combination thereof (T + D). The RNase protection assay is used to determine the mRNA expression of II-2 and TNF. (A), (C) relate to the induced expression and repression of II-2 mRNA and their quantification. (B), (D) relate to the induced expression and repression of IFN mRNA and their quantification, (con) means control and (n, d) undetectable.

Fig. 5 zeigt die Expression von Cytokin-mRNA. Makrophagen aus einer erfindungsgemäßen Maus bzw. einer Wildtyp-Maus werden mit Lipopolysaccharid (LPS) bzw. einer Kombination aus LPS und dex (L + D) behandelt. Mittels " RNase-protection assay" wird die - 7 - mRNA-Expression von TNFσ und 11-6 bestimmt. (A), (B), (C) betreffen die Expression und Reprimierung von 11-6 bzw. TNFσ mRNA und ihre Quantifizierung, (con) bedeutet Kontrolle.Figure 5 shows the expression of cytokine mRNA. Macrophages from a mouse according to the invention or a wild-type mouse are treated with lipopolysaccharide (LPS) or a combination of LPS and dex (L + D). With the "RNase-protection assay" the - 7 - mRNA expression of TNF σ and 11-6 determined. (A), (B), (C) relate to the expression and repression of 11-6 or TNF σ mRNA and their quantification, (con) means control.

Fig. 6 zeigt die Reaktion einer Maus bei einer Infektion bzw. einer Entzündung. (A) Eine erfindungsgemäße Maus bzw. eine Wildtyp-Maus wird mit LPS behandelt und die Serumkonzentration von TNF0 wird bestimmt. (B) Die Haut einer erfindungsgemäßen Maus bzw. einer Wildtyp-Maus wird mit TPA bzw. einer Kombination aus TPA und Dexamethason (T + D) behandelt und die Serumkonzentration von 11-6 wird bestimmt. (C) Das Ohr einer erfindungsgemäßen Maus bzw. einer Wildtyp-Maus wird mit TPA bzw. einer Kombination aus TPA und Dexamethason (T + D) behandelt und ein bestimmter Bereich des Ohres wird herausgeschnitten und sein Gewicht als Maß für ein erhaltenes Ödem bestimmt.Fig. 6 shows the response of a mouse to infection or inflammation. (A) A mouse according to the invention or a wild-type mouse is treated with LPS and the serum concentration of TNF 0 is determined. (B) The skin of a mouse according to the invention or a wild-type mouse is treated with TPA or a combination of TPA and dexamethasone (T + D) and the serum concentration of 11-6 is determined. (C) The ear of a mouse or a wild-type mouse according to the invention is treated with TPA or a combination of TPA and dexamethasone (T + D) and a certain area of the ear is cut out and its weight is determined as a measure of edema obtained.

Die Erfindung wird durch die nachstehenden Beispiele erläutertThe invention is illustrated by the examples below

Beispiel 1 : Bereitstellung eines erfindungsgemäßen nicht-menschlichen SäugetiersExample 1: Provision of a non-human mammal according to the invention

Es wird ein nicht-menschliches Säugetier bereitgestellt, dessen GR in seiner induzierenden Funktion ausgeschaltet ist. Hierzu wird ein Vektor verwendet, der ca. 1 1 kb des GR-Gens, einschließlich der Exons 3 und 4 aufweist. Ferner weist der Vektor in Exon 4 eine Punktmutation auf, wodurch der hierdurch kodierte GR an der Position 458 anstelle von Alanin Threonin aufweist. Desweiteren umfaßt der Vektor eine loxP-tkneo-Selektionscassette (vgl. Fig. 1 ) .A non-human mammal is provided whose GR is switched off in its inducing function. For this purpose, a vector is used which has approximately 1 1 kb of the GR gene, including exons 3 and 4. Furthermore, the vector in exon 4 has a point mutation, as a result of which the GR encoded thereby at position 458 has threonine instead of alanine. Furthermore, the vector comprises a loxP-tkneo selection cassette (see FIG. 1).

Dieser Vektor wird durch Elektroporation in die embryonalen Maus-Stammzellen E14/1 eingeführt. Stabil transfizierte Zellen werden durch Selektion mit 350 μg/ml G41 8 erhalten. Diese Zellen werden einer transienten Transfektion mit 20 μg eines Cre-Expressionsplasmids unterzogen, bevor ihnen 1 /M Gancyclovir - 8 - zugegeben wird. Damit wird auf Zellen selektioniert, welche die Selektions- cassette verloren haben. Diese Zellen weisen im GR-Gen die angegebene Punktmutation und zusätzlich ca. 50 Basenpaare entlang der verbliebenen lox P-Stelle im Intron 3 auf.This vector is introduced into the mouse embryonic stem cells E14 / 1 by electroporation. Stably transfected cells are obtained by selection with 350 μg / ml G41 8. These cells are transiently transfected with 20 μg of a Cre expression plasmid before being given 1 / M Gancyclovir - 8 - is added. This selects for cells that have lost the selection cassette. These cells have the specified point mutation in the GR gene and additionally about 50 base pairs along the remaining lox P site in intron 3.

Letztere Zellen werden in Maus-Blastozysten injiziert und diese dann in weibliche Mäuse eingeführt. Es werden Chimäre Mäuse erhalten, aus denen durch Verpaa- rung heterozygote und homozygote Mäuse gewonnen werden.The latter cells are injected into mouse blastocysts and then introduced into female mice. Chimeric mice are obtained, from which heterozygous and homozygous mice are obtained by mating.

Beispiel 2: Nachweis einzelner Funktionen eines erfindungsgemäßen nichtmenschlichen SäugetiersExample 2: Detection of individual functions of a non-human mammal according to the invention

Es wird das in Beispiel 1 bereitgestellte nicht-menschliche Säugetier verwendet. Dieses wird in Testversuche eingesetzt, die spezifisch für die einzelnen Funktionen von GR sind.The non-human mammal provided in Example 1 is used. This is used in test trials that are specific to the individual functions of GR.

(a) Induzierung von Enzymen der Glukoneogenese(a) Induction of enzymes of gluconeogenesis

Einer erfindungsgemäßen Maus und einer Wildtyp-Maus werden jeweils 10 yg/100 g Dexamethason bzw. PBS injiziert. Nach zwei Stunden werden die Tiere getötet. Leber-RNA wird isoliert und die mRNA-Expression von Tyrosinamino- transferase (TAT) bzw. von ß-Aktin als Kontrolle wird in einem Northern-Blot bestimmt (vgl. Fig. 2a).A mouse according to the invention and a wild-type mouse are each injected with 10 yg / 100 g dexamethasone or PBS. The animals are killed after two hours. Liver RNA is isolated and the mRNA expression of tyrosine amino transferase (TAT) or of β-actin as a control is determined in a Northern blot (cf. FIG. 2a).

Es zeigt sich, daß eine Wildtyp-Maus, nicht aber eine erfindungsgemäße Maus eine TAT mRNA induzieren kann.It is shown that a wild-type mouse, but not a mouse according to the invention, can induce a TAT mRNA.

(b) Induzierung der Proliferation von Erythrozyten-Vorläuferzellen(b) inducing the proliferation of erythrocyte progenitor cells

Aus einem erfindungsgemäßen Maus- und einem Wildtyp-Mausembryo wird jeweils fötale Leber isoliert und in Einzelzellen dispergiert. Diese werden dann in - 9 - modifiziertem CFU-E-Medium, das SCF, hEpo, IGF-1 und Dexamethason enthält, vermehrt. Eine Wachstumskinetik wird erstellt, indem täglich die Zellen unter Verwendung eines elektronischen Zellzählers gezählt werden (vgl. Fig. 2b) .In each case, fetal liver is isolated from a mouse and a wild-type mouse embryo according to the invention and dispersed in individual cells. These are then in - 9 - Modified CFU-E medium containing SCF, hEpo, IGF-1 and dexamethasone increased. A growth kinetics is created by counting the cells daily using an electronic cell counter (see FIG. 2b).

Es zeigt sich, daß eine Wildtyp-Maus, nicht aber eine erfindungsgemäße Maus die Proliferation von Erythrozyten-Vorläuferzellen induzieren kann.It is shown that a wild-type mouse, but not a mouse according to the invention, can induce the proliferation of erythrocyte precursor cells.

(c) Induzierung der Apoptose von Thymozyten(c) inducing apoptosis of thymocytes

Aus einer 6-1 2 Wochen alten erfindungsgemäßen Maus und einer entsprechend alten Wildtyp-Maus werden jeweils Thymozyten isoliert und in RPMI-Medium 24 Stunden in Gegenwart bzw. Abwesenheit von Dexamethason ( 10"6M) kultiviert. Die Zellen werden dann mit Propidiumjodid gefärbt und auf ihre Fluoreszenzintensität mittels FACS analysiert. Der Verlust an DNA-Gehalt wird als Maß für Apoptose genommen.Thymocytes are isolated from a 6-1 2 week old mouse according to the invention and a correspondingly old wild-type mouse and cultured in RPMI medium for 24 hours in the presence or absence of dexamethasone (10 "6 M). The cells are then stained with propidium iodide and analyzed for their fluorescence intensity using FACS. The loss of DNA content is taken as a measure of apoptosis.

Es zeigt sich, daß eine Wildtyp-Maus, nicht aber eine erfindungsgemäße Maus die Apoptose von Thymozyten induzieren kann.It is shown that a wild-type mouse, but not a mouse according to the invention, can induce the apoptosis of thymocytes.

(d) Inhibierung der AP-1 bedingten Aktivierung des Kollagenase-Gens(d) Inhibition of AP-1-induced activation of the collagenase gene

Primäre Fibroblasten werden aus Embryonen einer erfindungsgemäßen Maus und einer Wildtyp-Maus isoliert. Die Fibroblasten werden 6 Stunden mit 10"6M Dexamethason, 10"7 M TPA oder beidem behandelt, bevor RNA isoliert und die mRNA-Expression von MMP-1 3(Maus-Kollagenase 3-Gen) und von GAPDH als Kontrolle in einem Northern-Blot bestimmt werden (vgl. Fig. 3).Primary fibroblasts are isolated from embryos of a mouse according to the invention and a wild-type mouse. The fibroblasts are treated for 6 hours with 10 "6 M dexamethasone, 10 " 7 M TPA or both before RNA is isolated and the mRNA expression of MMP-1 3 (mouse collagenase 3 gene) and GAPDH as a control in a Northern -Blot can be determined (see FIG. 3).

Es zeigt sich, daß eine Wildtyp-Maus und auch eine erfindungsgemäße Maus die AP1 -bedingte Aktivierung des Kollagenase-Gens inhibieren können.It is shown that a wild-type mouse and also a mouse according to the invention can inhibit the AP1-related activation of the collagenase gene.

Somit wird deutlich, daß ein erfindungsgemäßes Säugetier, z.B. eine Maus, - 10 - einen GR exprimiert, bei dem die reprimierende Funktion erhalten, die induzierende Funktion allerdings verändert, insbesondere ausgeschaltet ist.It is thus clear that a mammal according to the invention, for example a mouse, - 10 - expresses a GR in which the repressing function is retained, but the inducing function is changed, in particular switched off.

Beispiel 3: Untersuchungen an einem erfindungsgemäßen nicht-menschlichen Säugetier bzw. Zellen davon hinsichtlich immunsuppressiver bzw. entzündungshemmender AktivitätExample 3: Investigations on a non-human mammal according to the invention or cells thereof with regard to immunosuppressive or anti-inflammatory activity

3.1 Untersuchungen an primären Thymozyten3.1 Investigations on primary thymocytes

Primäre Thymozyten aus einer erfindungsgemäßen Maus bzw. einer Wildtyp-Maus werden isoliert und 6 h mit 0,5μg/ml lonomycin und 10μg/ml Phorbolester (TPA) bzw. 10~6 M Dexamethason (dex) bzw. einer Kombination daraus (T + D) behandelt. 2μg Gesamt-RNA werden jeweils einem "RNase-protection assay" unterzogen.Primary thymocytes from a mouse according to the invention or a wild-type mouse are isolated and 6 h with 0.5μg / ml ionomycin and 10μg / ml phorbol ester (TPA) or 10 ~ 6 M dexamethasone (dex) or a combination thereof (T + D) treated. 2 μg total RNA are each subjected to an “RNase protection assay”.

Es zeigt sich, daß in primären Thymozyten beider Mäuse eine vergleichbare Expression von Cytokin-mRNA, z.B. II-2 und IFN.. bzw. eine vergleichbare Reprimierung erhalten wird.It turns out that in primary thymocytes of both mice a comparable expression of cytokine mRNA, e.g. II-2 and IFN .. or a comparable repression is obtained.

3.2 Untersuchungen an peritonealen Makrophagen3.2 Studies on peritoneal macrophages

Peritoneale Makrophagen aus einer erfindungsgemäßen Maus bzw. einer Wildtyp-Maus werden 5 d nach einer Thioglykolat-Behandlung isoliert und nach 24 h für 2 h mit 100 ng/ml Lipopolysaccharid (LPS) bzw. einer Kombination aus 10"6M Dexamethason und Lipopolysaccharid (L + D) behandelt. 0,5μg Gesamt-RNA wird jeweils einem "RNAse-Protection assay" unterzogen.Peritoneal macrophages from a mouse according to the invention or a wild-type mouse are isolated 5 d after a thioglycolate treatment and after 24 h for 2 h with 100 ng / ml lipopolysaccharide (LPS) or a combination of 10 "6 M dexamethasone and lipopolysaccharide ( L + D) 0.5 μg of total RNA is subjected to an "RNAse protection assay".

Es zeigt sich, daß in peritonealen Makrophagen beider Mäuse eine vergleichbare Expression von Cytokin-mRNA, z.B. TNFσ und II-6, bzw. deine vergleichbare Reprimierung erhalten wird. - 1 1 -It turns out that in peritoneal macrophages of both mice a comparable expression of cytokine mRNA, eg TNF σ and II-6, or your comparable repression is obtained. - 1 1 -

3.3 Untersuchungen an Mäusen3.3 Studies in mice

(A) Einer erfindungsgemäßen Maus bzw. einer Wildtyp-Maus werden jeweils 10Oμg LPS verabreicht. Die Mäuse werden zu den Zeitpunkten 0, 60 min und 1 80 min getötet. Serum wird jeweils isoliert und die TNFα-Konzentration durch einen üblichen ELISA Kit bestimmt.(A) A mouse according to the invention or a wild-type mouse are administered in each case 10 μg LPS. The mice are sacrificed at 0, 60 min and 1 80 min. Serum is isolated in each case and the TNF α concentration is determined using a conventional ELISA kit.

Es zeigt sich, daß die Induktion von TNFσ und seine Reprimierung in beiden Mäusen vergleichbar ist.It can be seen that the induction of TNF σ and its repression are comparable in both mice.

(B) Die Rückenhaut einer erfindungsgemäßen Maus bzw. einer Wildtyp- Maus wird jeweils mit TPA bzw. einer Kombination aus TPA und Dexamethason (T + D) behandelt. Serum wird jeweils isoliert und die ll-6-Konzentration durch einen üblichen ELISA bestimmt.(B) The back skin of a mouse according to the invention or a wild-type mouse is in each case treated with TPA or a combination of TPA and dexamethasone (T + D). Serum is isolated in each case and the II-6 concentration is determined by a conventional ELISA.

Es zeigt sich, daß die Induktion von II-6 sowie ihre Reprimierung in beiden Mäusen vergleichbar ist.It can be seen that the induction of II-6 and its repression are comparable in both mice.

(C) Das linke Ohr einer erfindungsgemäßen Maus bzw. einer Wildtyp- Maus wird jeweils mit TPA bzw. einer Kombination aus TPA und Dexamethason (T + D) behandelt. Ein bestimmter Bereich des Ohres wird jeweils entfernt und das Gewicht des gebildeten Ödems bestimmt.(C) The left ear of a mouse according to the invention or a wild-type mouse is in each case treated with TPA or a combination of TPA and dexamethasone (T + D). A certain area of the ear is removed and the weight of the edema formed is determined.

Es zeigt sich, daß das Gewicht des Ödems nach der jeweiligen Behandlung in beiden Mäusen vergleichbar ist.It turns out that the weight of the edema after the respective treatment is comparable in both mice.

Somit wird aufgezeigt, daß eine erfindungsgemäße Maus und deren Zellen immunsuppressive und entzündungshemmende Aktivität aufweisen. Diese ist mit jener einer Wildtyp-Maus vergleichbar. It is thus shown that a mouse according to the invention and its cells have immunosuppressive and anti-inflammatory activity. This is comparable to that of a wild-type mouse.

Claims

- 1 2 -Patentansprüche - 1 2 -patent claims 1 . Nicht-menschliches Säugetier, dessen Glukokortikoid-Rezeptor (GR) in seiner induzierenden Funktion verändert ist.1 . Non-human mammal whose glucocorticoid receptor (GR) is altered in its inducing function. 2. Nicht-menschliches Säugetier nach Anspruch 1 , wobei die induzierende Funktion ausgeschaltet ist.2. The non-human mammal of claim 1, wherein the inducing function is turned off. 3. Nicht-menschliches Säugetier nach Anspruch 1 oder 2, wobei GR in seiner Dimerisierung bzw. DNA-Bindung mutiert ist.3. Non-human mammal according to claim 1 or 2, wherein GR is mutated in its dimerization or DNA binding. 4. Nicht-menschliches Säugetier nach einem der Ansprüche 1 -3, wobei GR an der Position 458 ein Threonin aufweist.4. A non-human mammal according to any one of claims 1-3, wherein GR at position 458 has a threonine. 5. Verfahren zur Bereitstellung eines nicht-menschlichen Säugetiers nach einem der Ansprüche 1 -4, umfassend die folgenden Schritte:5. A method of providing a non-human mammal according to any one of claims 1-4, comprising the following steps: (a) Transfektion von embryonalen Stammzellen eines nicht-menschlichen Säugetiers mit einem Vektor, der eine Rekombination zwischen der für die induzierende Funktion von GR kodierenden DNA der embryonalen Stammzellen und einer entsprechenden mutierten DNA des Vektors ermöglicht,(a) transfection of embryonic stem cells of a non-human mammal with a vector which enables a recombination between the DNA of the embryonic stem cells coding for the inducing function of GR and a corresponding mutated DNA of the vector, (b) Isolierung von in (a) stabil transfizierten Zellen und Einbringung dieser in weibliche Tiere eines nicht-menschlichen Säugetiers, sowie(b) isolation of cells stably transfected in (a) and introduction into female animals of a non-human mammal, and (c) Analyse der in (b) erhaltenen Nachkommen auf einen GR, dessen induzierende Funktion verändert ist.(c) Analysis of the offspring obtained in (b) for a GR whose inducing function is changed. 6. Verfahren nach Anspruch 5, wobei die induzierende Funktion ausgeschaltet ist. - 1 3 -6. The method of claim 5, wherein the inducing function is switched off. - 1 3 - 7. Verfahren nach Anspruch 6, wobei GR in seiner Dimerisierung bzw. DNA- Bindung mutiert ist.7. The method according to claim 6, wherein GR is mutated in its dimerization or DNA binding. 8. Verfahren nach einem der Ansprüche 5-7, wobei GR an der Position 458 ein Threonin aufweist.8. The method according to any one of claims 5-7, wherein GR has a threonine at position 458. 9. Verwendung eines nicht-menschlichen Säugetiers nach einem der Ansprüche 1 -4 zur Testung von Substanzen, Arzneimitteln und/oder Therapieansätzen hinsichtlich der reprimierenden Funktion von GR.9. Use of a non-human mammal according to one of claims 1 -4 for testing substances, drugs and / or therapeutic approaches with regard to the repressing function of GR. 10. Verwendung nach Anspruch 9, wobei die Substanzen, Arzneimittel und/- oder Therapieansätze hinsichtlich Störungen der Akut-Phase-Reaktion, eines septischen Schocks, Asthma, eines akuten Atemnot-Syndroms, inflammatorischer Erkrankungen, Autoimmunerkrankungen, wie rheuma- toide Arthritis oder multiple Sklerose, neuropsychiatrischen Erkrankungen,10. Use according to claim 9, wherein the substances, drugs and / or therapeutic approaches with regard to disorders of the acute phase reaction, septic shock, asthma, acute shortness of breath syndrome, inflammatory diseases, autoimmune diseases, such as rheumatoid arthritis or multiple Sclerosis, neuropsychiatric disorders, Erkrankungen der Hypothalamus-Hypophysen-Nebennieren-Achse,Osteo- porose, Diabetes, Steroid-Myopathie oder Hauterkrankungen zu verstehen sind. Diseases of the hypothalamic-pituitary-adrenal axis, osteoporosis, diabetes, steroid myopathy or skin disorders are to be understood.
PCT/DE1999/000747 1998-03-12 1999-03-12 Non-human transgenic mammal, method for producing same and its use Ceased WO1999045767A1 (en)

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WO1990007517A1 (en) * 1988-12-23 1990-07-12 The Salk Institute For Biological Studies Receptor transcription-repression activity compositions and methods

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