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WO1999045767A1 - Mammifere transgenique non humain, procede permettant de le preparer et utilisation - Google Patents

Mammifere transgenique non humain, procede permettant de le preparer et utilisation 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
Application number
PCT/DE1999/000747
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German (de)
English (en)
Inventor
Holger Reichardt
Günther Schütz
Klaus KÄSTNER
Peter Herrlich
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Deutsches Krebsforschungszentrum DKFZ
Karlsruher Institut fuer Technologie KIT
Original Assignee
Deutsches Krebsforschungszentrum DKFZ
Forschungszentrum Karlsruhe GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Deutsches Krebsforschungszentrum DKFZ, Forschungszentrum Karlsruhe GmbH filed Critical Deutsches Krebsforschungszentrum DKFZ
Priority to JP2000535196A priority Critical patent/JP2002505858A/ja
Priority to AU39249/99A priority patent/AU3924999A/en
Priority to EP99922034A priority patent/EP1061798A1/fr
Publication of WO1999045767A1 publication Critical patent/WO1999045767A1/fr
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

L'invention concerne un mammifère non humain dont la fonction inductrice du récepteur de glucocorticoïde (GR) est modifiée. L'invention concerne en outre un procédé permettant de préparer un mammifère de ce type et son utilisation pour tester des produits chimiques, des médicaments et des préparations thérapeutiques.
PCT/DE1999/000747 1998-03-12 1999-03-12 Mammifere transgenique non humain, procede permettant de le preparer et utilisation Ceased WO1999045767A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP2000535196A JP2002505858A (ja) 1998-03-12 1999-03-12 非ヒトトランスジェニック哺乳動物、その生産方法およびその使用
AU39249/99A AU3924999A (en) 1998-03-12 1999-03-12 Non-human transgenic mammal, method for producing same and its use
EP99922034A EP1061798A1 (fr) 1998-03-12 1999-03-12 Mammifere transgenique non humain, procede permettant de le preparer et utilisation

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE1998115958 DE19815958C1 (de) 1998-03-12 1998-03-12 Nicht-menschliches Säugetier sowie Verfahren zu seiner Herstellung und seine Verwendung
DE19815958.7 1998-03-12

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US09623991 A-371-Of-International 2001-02-06
US10/326,087 Continuation US20030172393A1 (en) 1998-03-12 2002-12-23 Non-human transgenic mammal, method for producing same and its use

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WO1999045767A1 true WO1999045767A1 (fr) 1999-09-16

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EP (1) EP1061798A1 (fr)
JP (1) JP2002505858A (fr)
AU (1) AU3924999A (fr)
DE (1) DE19815958C1 (fr)
WO (1) WO1999045767A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19933075C2 (de) * 1999-07-19 2001-11-29 Deutsches Krebsforsch Nicht-menschliches Säugetier mit gewebespezifisch verändertem Glukokortikoidrezeptor
DE10001975A1 (de) * 2000-01-18 2001-07-26 Deutsches Krebsforsch Nicht-menschliches Säugetier mit verändertem Glukokortikoid-Rezeptor und verändertem c-Fos

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990007517A1 (fr) * 1988-12-23 1990-07-12 The Salk Institute For Biological Studies Compositions a activite transcripto-repressive pour des recepteurs et procedes correspondants

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990007517A1 (fr) * 1988-12-23 1990-07-12 The Salk Institute For Biological Studies Compositions a activite transcripto-repressive pour des recepteurs et procedes correspondants

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
COLE T.J. ET AL.: "Targeted disruption of the glucocorticoid receptor gene blocks chromaffin cell development and severely retards lung maturation", GENES & DEVELOP., vol. 9, 1995, pages 1608 - 1621, XP002109180 *
HECK S. ET AL.: "A distinct modulating domain in glucocorticoid receptor monomers in the repression activity of the transcription factor AP-1", EMBO J., vol. 13, no. 17, 1994, pages 4087 - 4095, XP002109177 *
REICHARDT H. ET AL.: "DNA binding of the glucocorticoid receptor is not essential for survival.", CELL, vol. 93, 15 May 1998 (1998-05-15), pages 531 - 541, XP002109178 *
ZANDI E. ET AL.: "Zinc finger mutations that alter domain interactions in the glucocorticoid receptor", J. MOL. BIOL., vol. 230, 1993, pages 124 - 136, XP002109179 *

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EP1061798A1 (fr) 2000-12-27
AU3924999A (en) 1999-09-27
JP2002505858A (ja) 2002-02-26
DE19815958C1 (de) 1999-09-16

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