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WO2002033103A2 - Mammifere non humain presentant un recepteur de type i du pacap modifie - Google Patents

Mammifere non humain presentant un recepteur de type i du pacap modifie Download PDF

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WO2002033103A2
WO2002033103A2 PCT/DE2001/003957 DE0103957W WO0233103A2 WO 2002033103 A2 WO2002033103 A2 WO 2002033103A2 DE 0103957 W DE0103957 W DE 0103957W WO 0233103 A2 WO0233103 A2 WO 0233103A2
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receptor
human mammal
pacap type
mice
pacap
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WO2002033103A3 (fr
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Christiane Otto
Günther Schütz
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Deutsches Krebsforschungszentrum
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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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • 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
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • 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
    • 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
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0356—Animal model for processes and diseases of the central nervous system, e.g. stress, learning, schizophrenia, pain, epilepsy
    • 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

  • Non-human mammal with an altered PACAP type I receptor is an altered PACAP type I receptor
  • the present invention relates to a non-human mammal which has an altered, preferably inactivated, PACAP type I receptor (PACl).
  • PACl PACAP type I receptor
  • the present invention further relates to methods for producing such a mammal and its use for characterizing genes and / or testing substances, drugs and therapeutic approaches with regard to various diseases, such as e.g. visceral pain, learning disabilities, anxiety or dilated cardiomyopathy.
  • the PACAP type I receptor is a G protein-coupled receptor that binds the highly conserved neuropeptide PACAP ("pituitary adenylate cyclase activating polypeptide") with a thousand times higher affinity than the related peptide VIP.
  • Signal transmission mediated by the PACAP Type I receptor is believed to be involved in a wide range of physiological processes including neurotransmitters / neurotrophic actions, neuronal differentiation, synaptic plasticity and fertility.
  • the present invention is therefore based on the technical problem of providing means which allow the identification of genes which are involved in the development of diseases which are associated with impaired learning behavior, e.g. Learning deficiency, associated with pain, excessive fear behavior or dilated cardiomyopathy, and are therefore of therapeutic benefit, since the desired therapeutic effect can be achieved by influencing the expression of these genes.
  • impaired learning behavior e.g. Learning deficiency, associated with pain, excessive fear behavior or dilated cardiomyopathy
  • the gene coding for the PACAP type I receptor is a gene which is very well suited for the above purposes. Therefore, the problems discussed above can be addressed by providing a model system by creating animals, eg mice, in which the gene coding for the PACAP type I receptor is changed or inactivated.
  • animals eg mice
  • the gene coding for the PACAP type I receptor is changed or inactivated.
  • two different mouse mutants were generated, which have ubiquitous inactivation and tissue-specific inactivation of the PACAP type I receptor.
  • the Cre loxP system was used and exon 11 of the PACl gene was flanked with loxP sites. Mice with ubiquitous inactivation of the PACAP type I receptor showed reduced anxiety and increased locomotion. In addition, these animals showed a specific deficit in the hippocampus-dependent association learning.
  • the knockout mice according to the invention allow the search for genes which are regulated via the PACAP type I receptor and whose misregulation in the PACAP type I receptor-deficient mice is the reason for the deficits described above.
  • these knockout mice enable the identification of new genes that are involved in processes such as learning, pain, anxiety and dilated cardiomyopathy, and which in turn can be the starting point for the development of new drugs, e.g. anxiolytics or memory-enhancing therapeutic agents.
  • the PACAP type I receptor is particularly important for the transmission of visceral pain, but not of somatic pain, very selective therapeutic agents, preferably for the treatment of tumor pain, the side effects of which may well be below those of broader-acting anaesthetics.
  • the present invention thus relates to a non-human mammal in which the PACAP type I receptor (PACl) is ubiquitously or tissue-specifically altered, preferably inactivated.
  • Characteristic of this mammal with an altered PACAP type I receptor is a combination of two genetic manipulations: (a) insertion of two recognition sites for a recombinase into the gene coding for PACl in such a way that its function is not impaired, but the Excision of the information between the recognition parts leads to expression of an inactive PACl and (b) insertion of a gene which codes for a recombinase which recognizes the above recognition sites and is under the control of a tissue-specific promoter.
  • the procedure described above can also be used for the production of animals with a ubiquitously modified gene, the gene coding for the recombinase being under the control of a non-tissue-specific promoter.
  • non-human mammal encompasses any mammal whose PACl can be ubiquitously or tissue-specifically modified, preferably inactivated, for example mouse, rat, rabbit, horse, cattle, sheep, goat, monkey, pig, dog and cat, mouse is preferred.
  • PACAP type I receptor encompasses any PACAP type I receptor.
  • a non-human mammal according to the invention can be produced, for example, by the methods described in the examples below.
  • the inactivation of the PACAP type I receptor can be achieved, for example, by changing the coding gene in such a way that a PACAP type I receptor is expressed in which the ligand-binding domain is inactivated or one or more transmembranes Domains are deleted.
  • a transmembrane domain or a portion thereof is deleted, most preferably a deletion of exon 11 which results in the deletion of most of the transmembrane domain IV of the receptor.
  • the tissue-specific change / inactivation of the PACAP type I receptor is not restricted to specific tissues and can ultimately affect all tissues for which corresponding tissue-specific promoters are available.
  • the function of the PACAP type I receptor is preferably inactivated in the brain, for example by using a recombinase under the control of the Nestin promoter, even more preferred is the forebrain-specific inactivation, for example by using a under the control of the CaMKIi ⁇ promoter (Kellendonk et al., J. Mol. Biol. 285 (1999), 175-182) standing recombinase can be achieved.
  • a non-human mammal according to the invention can preferably be obtained by a method, the following
  • Steps include:
  • step (b) isolation of cells stably transfected in step (a) and introduction into a first non-human mammal;
  • step (d) introducing the injected oocytes from step (c) into a second non-human mammal;
  • step (f) Selection of the progeny obtained after step (e) for those with a tissue-specifically modified, in particular inactivated PACAP type I receptor.
  • embryonic stem cells used here includes all embryonic stem cells of a non-human mammal that are suitable for mutating the DNA coding for the PACAP type I receptor.
  • the embryonic stem cells are preferably from the mouse, in particular E14 / 1 cells.
  • vector refers to any vector which, after recombination with the genome of the non-human mammal, results in the insertion of two recognition sites for a recombinase into the DNA coding for the PACAP type I receptor, with regard to the function / Localization of the recognition sites apply the information given above, or (b) the integration of a coding for a recombinase, under the control of a (tissue-specific) promoter standing gene allowed into the genome, the recognition sites of the vector from (a) must be compatible with the recombinase of the vector from (b), ie recognized by this.
  • Tissue-specific elimination of the PACAP type I promoter can be achieved, for example, by inserting the two recognition sites for the recombinase into the genome in such a way that the corresponding recombinase under the control of a tissue-specific promoter inactivates the areas of the receptor discussed above or deleted. It is also advantageous if the vector has a marker with which selection can be made for stem cells in which the desired recombination has taken place.
  • One such marker is the loxP / tkneo cassette, which can be removed from the genome again using the Cre / loxP system.
  • step (a) the person skilled in the art knows conditions and materials in order to carry out steps (a) to (f) of the above method. For example, he will inject the cells stably transfected in step (a) into blastocysts of a mammalian donor female. The blastocysts are then introduced into sham-pregnant mammals. Some of the transferred blastocysts develop into mouse chimeras, some of which carry a modified PACAP type I receptor allele in the germ cells. Appropriate crossings then give rise to hetero- and homozygous mammals.
  • mammals By mating these mammals and crossing with mammals encoding a recombinase under the control of a tissue-specific promoter, mammals are obtained that are homozygous for the modified PACAP type I receptor allele and heterozygous for the recombinase gene.
  • the person skilled in the art will apply methods in which it can be demonstrated in the desired tissues that the receptor is modified or inactivated with regard to its biological activity, for example by determining to what extent the induction of PACAP type I -Receptor-dependent genes are weakened or prevented.
  • the choice of the promoter controlling the recombinase depends on the tissue in which the biological function of the PACAP type I receptor in the non-human mammal according to the invention is to be changed, preferably inactivated. Suitable promoters are known to the person skilled in the art. Such are for example for the nervous system (neurons and glial cells) as Nestin-Cre in Zimmermann et al. , Neuron 12.
  • any recognition site / recombinase system can be used which allows the effective excision of the gene or gene segment lying between the recognition sites for the recombinase.
  • Suitable systems are known to the person skilled in the art. These include e.g. the LoxP / Cre recombinase system or the FRT / FLP recombinase system (Sauer and Hendersen, Proc. Natl. Acad. Sci. 85 (1988), 5166-5170), the LoxP / Cre system being preferred. It is particularly preferred if the LoxP sites flank exon 11 of the gene coding for the PACAP type I receptor.
  • a non-human mammal according to the invention in which the PACAP type I receptor is ubiquitously changed / inactivated. This can be achieved by the recombinase being under the control of a non-tissue-specific promoter; see also the explanations in Example 1 below.
  • a non-human mammal according to the invention with a ubiquitously modified PACAP type I receptor can also be obtained by a process which comprises the following steps: (a) transfecting embryonic stem cells of a non-human mammal with a vector which, after homologous recombination with the genome of the non-human mammal, leads to the generation of a gene coding for an altered or inactivated PACAP type I receptor;
  • step (b) isolating the cells obtained in step (a) and introducing them into blastocysts;
  • step (c) introducing the injected blastocysts from step (b) into a non-human mammal;
  • step (d) Examination of the non-human mammal obtained after step (c) for a ubiquitously modified, in particular inactivated, PACAP type I receptor.
  • the non-human mammal it is possible to investigate the effects of, for example tissue-specific, change or inactivation of the PACAP type I receptor, for example with regard to the change in the expression of specific genes and / or the associated phenotypic peculiarities.
  • tissue-specific, change or inactivation of the PACAP type I receptor for example with regard to the change in the expression of specific genes and / or the associated phenotypic peculiarities.
  • Such inactivation of the PACAP-type receptor also leads to dilated cardiomyopathy.
  • the forebrain-specific inactivation leads to a deficit in the hippocampus-dependent association learning.
  • a comparison of gene expression in animals in which the PACAP type I receptor was ubiquitously inactivated or only in the forebrain with control animals thus allows the identification of genes that are involved in processes such as learning, pain, anxiety or dilated cardiomyopathy. are involved and can in turn be the starting point for the development of new medicines, e.g. anxiolytics or memory-enhancing therapeutic agents.
  • new medicines e.g. anxiolytics or memory-enhancing therapeutic agents.
  • the knowledge of these genes and their function then allows the development of new approaches for pharmacological Influencing the processes described above, for example by providing compounds which specifically influence the expression of these genes or the activity of the gene products.
  • the non-human mammal not only can genes be identified which are under the control of the PACAP type I receptor, but also their function or the possibly pathogenic conditions triggered by a malfunction of the function can be determined. Since the PACAP type I receptor is particularly important for the transmission of visceral pain, but not of somatic pain, very selective therapeutic agents, preferably for the treatment of tumor pain, can be developed, the side effects of which are likely to be significantly lower than those of broader-acting analgesics ,
  • the present invention preferably also relates to the use of the non-human mammal to characterize genes and / or test substances, drugs and / or therapeutic approaches that have an impact on processes related to learning ability, pain, anxiety behavior and dilated cardiomyopathy these processes are diseases or related symptoms such as visceral pain, learning or memory impairment, anxiety or dilated cardiomyopathy.
  • PACAP type II receptors (VPACl and VPAC2) are not upregulated in PACl "7" brains (M: lkb ladder; GAPDH: glyceraldehyde-3-phosphate dehydrogenase).
  • mice showed increased horizontal (p ⁇ 0.01) and vertical locomotor activity (p ⁇ 0.01). The results of the first day of observation are shown.
  • mice had dilated cardiomyopathy, which affected all 4 ventricles. These mice also showed centroacinous fatty liver.
  • mice showed altered gene expression.
  • ANP atrial natriuret. Factor
  • SERCA sarcoplasmatic calcium ATPase
  • Inner pri er 1 5 '- T C T A G A A T A A C T T C G T A T A
  • Interior primer 2 5 '- AT AAC T T G C TA TAG CATAC
  • the 5 'homology arm a 3.5 kbp Spel / BamHI fragment, which carries exons 7 to 10 of the PACl gene (all DNA fragments used come from a genomic ⁇ phage 18, which is from a genomic library was isolated (Kastner et al., Genomics 20 (1994), 377-385)), was subcloned into the "Bluescript” vector Bluescript IIKS + " (Stratagene) and fused with the 0.35 kbp BamHI / HindIII fragment , the "includes the upstream loxP site and exon.
  • G418-resistant clones were analyzed by Southern blot using probes outside the homology arms.
  • the 5 'probe represents a 500 bp Pstl-Spel fragment which connects directly to the 5' homology arm.
  • the 3 'probe is a 350 bg HindIII fragment which connects directly to the 3' homology arm. The frequency of homologous recombination was 12%.
  • Homologously recombined clones were transiently transfected with a Cre expression plasmid pHD2 (provided by F. Weih, DKFZ, Heidelberg, Germany) (20 ⁇ g) and subclones were selected in the presence of gancyclovir (1 ⁇ M). Which the PAC 1 _ - or PACL CaMKCre2 allele bearing mice were analyzed by blastocyst injection (Hogan et al, Manipulating the Mouse Embryo; Cold Spring Harbor Laboratory Press., 1994) were obtained.
  • nlsCre was cloned into a CaMKIl vector as described recently ⁇ .Kellendonk et al., J. Mol. Biol. 285 (1999), 175-182).
  • Linearized pMM403-Cre insertion DNA was injected into the pronuclei of C57BI / 6 oocytes (Kellendonk et al., J. Mol. Biol. 285 (1999), 175-182) and various transgenic lines were obtained.
  • the expression pattern of the Cre recombinase was defined using an anti-Cre recombinase antibody (Kellendonk et al., J. Mol. Biol.
  • RNase protection analyzes and in situ hybridization were carried out as described recently (Otto et al., Mol. Brain Res. .66
  • mice in three different laboratories / countries were unt he seeks.
  • the results were highly reproducible and were analyzed using the Student T-Test and ANOVA. The results are presented as mean values ⁇ standard deviation. To directly compare the two mutant mouse strains, all experiments in this study were carried out with the same genetic background (75% C75BI / 6/25% 129 Ola).
  • Example 2 Studies on the lack of expression of PACl in the mutant mice obtained in Example 1
  • mice were produced according to Example 1 above.
  • Exon 11 which codes for most of the transmembrane domain IV of the receptor protein, was used to inactivate all previously known splice variants of PACl inactivated.
  • ES embryonic stem cells
  • Two different PACl alleles were generated ( Figure la).
  • the PAC1 " allele with missing exon 11 was injected into blastocysts to produce PACl " / _ mice with ubiquitous inactivation of PACl.
  • exon 11 was flanked by two loxP recognition sites for the recombinase-controlled excision of the DNA sequence in between (FIG. 1 a).
  • PACl loxP is thus inactivated in every cell that expresses the recombinase.
  • PACl loxP homozygous mice appear normal and the expression of the PACl mRNA is identical to that of Wildty mice.
  • PACl loxP mice were crossed with transgenic mice (CaMKCre mice), the Cre recombinase expressing under the control of the CaMotKI promoter ,
  • PACl CaMKCre mice show, according to the expression pattern for Cre recombinase, inactivation of PACl mainly in three brain areas, the olfactory bulb, the cortex areas of the forebrain and the toothed gyrus ( Figure If).
  • PAC1 "- mice showed ubiquitous inactivation of PACl. Wild-type transcripts of PACl are completely missing (FIG. 1b, e). Instead, an alternatively fed transcript is detectable in PAC1 " / - brains, which 8% of the Wild-type RNA level reached ( Figure lb).
  • mice as well as PAC CaMKCre2 mice are the serrated gyrus and neocortex areas of the forebrain. Since lesions of the neocortex did not affect the contextual anxiety conditioning, the presynaptic PAC1-mediated signal transmission at the "mossy fiber" synapse seems to play a critical role for the consolidation of the contextual anxiety in long-term memory. In contrast to pvc CaMKCre2 mice, PAC "/ _ mice were deficient in the hippocampus-dependent short-term test regarding the conditioning of contextual anxiety (Figure 3b). This finding is consistent with the previous and ubiquitous inactivation of PACl in these mutants.
  • mice 7- mice were crossed back into the C57bl6 background and phenotypic and genotypic analysis of the mice was performed. It was found that 90% of the mice died on day 8-14. Furthermore, the mice showed progressive weakness and absence the weight gain from day 6 after birth ( Figure 4 (a)) and the mice also showed dilated cardiomyopathy affecting all 4 ventricles. The mice also had a suitable centroacinar fatty liver, as would be expected in hypoxemia, which is due to the reduced pumping power of the heart (FIG. 4 (b)). At the molecular level, a change in gene expression with respect to ANP (up-regulation) and SERCA (under-regulation) observed.
  • ANP up-regulation
  • SERCA under-regulation

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Abstract

L'invention concerne un mammifère non humain qui présente un récepteur de type I du PACAP (PAC1) modifié, de préférence inactivé. L'invention concerne en outre un procédé permettant d'obtenir un tel mammifère, ainsi que l'utilisation de ce dernier pour la caractérisation de gènes et/ou l'essai de substances, de médicaments et de préparations thérapeutiques pour le traitement de maladies telles que les douleurs viscérales, la difficulté à apprendre, les états d'anxiété ou la cardiomyopathie dilatée.
PCT/DE2001/003957 2000-10-16 2001-10-16 Mammifere non humain presentant un recepteur de type i du pacap modifie WO2002033103A2 (fr)

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
WO2009033489A3 (fr) * 2007-09-11 2009-05-07 Glostrup Hospital Traitement de la migraine et de céphalées par des inhibiteurs sélectifs de pac1

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