WO1996034094A1 - Non-human transgenic animal with modified d2 receptor expression - Google Patents
Non-human transgenic animal with modified d2 receptor expression Download PDFInfo
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- WO1996034094A1 WO1996034094A1 PCT/FR1996/000624 FR9600624W WO9634094A1 WO 1996034094 A1 WO1996034094 A1 WO 1996034094A1 FR 9600624 W FR9600624 W FR 9600624W WO 9634094 A1 WO9634094 A1 WO 9634094A1
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- C12N15/09—Recombinant DNA-technology
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- 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
- C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
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- A01K2267/0318—Animal model for neurodegenerative disease, e.g. non- Alzheimer's
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- 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
Definitions
- the subject of the invention is a transgenic or human animal in which the expression of the gene coding for the D 2 receptor is modified.
- Dopamine is a neurotransmitter that regulates many physiological functions. In the central nervous system, dopamine is involved in the coordination of movement, visual perception, knowledge, emotion and in neuroendocrine control of the pituitary gland
- Dopamine activation of membrane receptors generates the formation of intracellular secondary messengers.
- Dopamine receptors belong to the family of receptors with seven transmembrane domains coupled to G proteins of the 7TM domain (JA Gingrich and MG Caron, Annu. Rev. Neurosci., 16: 299 (1993)).
- Pharmacological and biochemical studies have described the existence of two classes of dopamine receptors Dj and D 2 .
- the Dj receptor has been identified as an activator of adenylate cyclase (AC), unlike D 2 which is described as an inhibitor of the same cellular effector.
- AC adenylate cyclase
- D2 receptor cDNA JR Bunzow et al., Nature, 336: 783 (1988)
- D3 P. Sokoloff et al., Nature, 347: 146 (1990)
- D 4 HMM Van Toi et al., Nature, 350: 610 (1991)
- Dj A. Dearry et al., Nature, 347: 72 (1990)
- D5 DK Grandy et al., Proc. Natl. Acad. Sci. USA, 88: 9175
- Dj and D5 receptors are stimulators of adenylate cyclase.
- the D5 receptor has a higher affinity for dopamine than D1. The pattern is less clear for the group of D-type receptors. Now the mechanism by which the D3 and D4 receptors transduce the signal, remains uncertain, although they have a pharmacological profile close to D.
- D3 A role for D3 as an autoreceptor has been proposed on the basis of a higher affinity for dopamine, in comparison with D 2 (JA Gingrich and MG Caron, Annu. Rev. Neurosci., 16: 299 (1993)).
- the D4 receptor has a greater affinity (about ten times) than D 2 and D3 for clozapine, an antipsychotic, at concentrations that are active in vivo, and it has been proposed as a target for this drug.
- the absence of identification of a transduction pathway for these receptors could be due to the absence of specific G proteins in the cell lines tested.
- the entire dopamine receptor subfamily is widely expressed in the central nervous system, particularly in the caudate-putamen, nucleus accumbens, hypothalamus and olfactory tubers.
- the pharmacological characteristics common between the D ⁇ and D groups of dopaminergic receptors are also reflected at the amino acid level where a homology of more than 40% is observed on the total sequence between the members of each group.
- the first isolated D 2 receptor cDNA codes for a protein of 415 amino acids and was isolated from a rat brain cDNA library (JR Bunzow et al., Nature, 336: 783 (1988)) .
- the pharmacological profile obtained from transfected cells clearly identifies it as a dopamine D 2 receptor.
- another cDNA was isolated from different sources which is 100% homologous to the first, except for insertion of 29 amino acids into the third intracellular domain of the receptor.
- This cDNA codes for a protein of 444 amino acids, which has been found in rats, mice, cattle, and humans (human cDNA has 445 amino acids). (JA Gingrich and MG Caron, Annu. Rev.
- Genomic DNA analyzes have shown that the two cDNAs are products of the same gene generated by an alternative splicing mechanism that adds an 87 base pair exon to the larger of the messenger RNAs. These receptors have been designated by DS for the shortest form, and by D 2 L for the longest form.
- the structure of the genomic fragment containing the D 2 gene has been determined. Eight exons were found in all the species analyzed, with a surprising characteristic represented by the presence of a very large intron located upstream of the first coding exon. The presence of such a large intronic sequence corresponds to a mechanism for controlling the expression of the D gene. Cloning of the D gene promoter region has been reported; since no characteristic trait has been described, it seems to correspond to the characteristics of a ubiquitous promoter. Interestingly, the two isoforms of the D 2 receptor are co-expressed in all of the tissues tested with a ratio which normally favor the expression of D 2 L. (J.-P. Montmayeur et al., Febs lett., 278 : 239 (1991)).
- these two isoforms have similar profiles and bind to the spiperone antagonist with similar affinity.
- the activation of these two D 2 receptors mainly results in the inhibition of adenylate cyclase. However, it appears that they are also capable of activating other signal transduction pathways. (JA Gingrich and MG Caron, Annu. Rev.
- One aspect of the The invention is to provide non-human transgenic mammalian animals capable of overexpressing one and / or the other isoform of the D 2 receptor.
- One of the other aspects of the invention is to provide non-human transgenic mammalian animals in which either the D 2 L isoform is no longer expressed, or the two D 2 L and D 2 S isoforms are no longer expressed.
- One of the other aspects of the invention is to provide an animal model capable of screening drugs which are active on pathologies involving the D 2 receptors.
- the subject of the invention is the use of a non-human transgenic mammalian animal whose expression of the gene coding for the dopaminergic receptor D 2 is modified, in particular on the lactotropic cells of the pituitary gland, compared to a normal expression, in particular on the lactotropic cells of the pituitary gland, for the determination of a drug which is active on pathologies involving the D 2 receptor.
- the term "mammal” includes all mammals except humans, advantageously rodents and in particular mice.
- the normal expression of the dopaminergic receptor D 2 can be defined by several methods:
- RNAs are separated on a denaturing agarose gel by electrophoresis, then the RNAs are transferred and fixed on a nitrocellulose or nylon type membrane.
- a probe corresponding to the entire cDNA of the gene we uses a probe corresponding to the entire cDNA of the gene. (The D 2 L or D 2 S cDNA can be used interchangeably).
- the RNAse enzyme protection method is used.
- the 370 base pair AccI fragment was cloned (positions of AccI sites: 700 and
- this plasmid is then linearized by cleavage with the enzyme Bsu36I and transcribed in vitro using the T3 polymerase using nucleotides labeled with 32 p s or having a labeled RNA probe.
- the size of this probe is 300 nucleotides.
- the protected fragments are 202 nucleotides for DL and 130 nucleotides for D 2 S. (See FIGS. 7 or 8).
- This probe is used for hybridization with RNA from regions expressing the D receptor (striatum, pituitary gland).
- the hybrids are then digested with the RNAse enzyme and two fragments are obtained: one corresponding to D 2 L of 202 base pairs, and the other corresponding to DS of 130 base pairs.
- This analysis is called quantitative because it makes it possible to evaluate exactly the proportion of RNA corresponding to one or the other of the isoforms. To determine if there is a change in the expression of the D gene, one can proceed as indicated below.
- the modification of the expression of the gene coding for the dopaminergic receptor D 2 corresponds either to an overexpression of the gene, in particular on the pituitary cells of the pituitary gland, compared to the normal expression defined above, that is to say the absence of expression of the D 2 gene.
- the overexpression of D 2 can be determined as follows.
- transgene is defined as a fragment of DNA which is inserted artificially (i.e. other than by sexual reproduction) in a cell and which becomes part of the genome of the animal which develops from that cell, or that is modified from the DNA normally present in the cell.
- a transgene may be a gene homologous to a natural gene of the transgenic animal but which is inserted into the genome of the animal at a position which differs from that of the natural homolog.
- the DNA extracted from a sample for example from a mouse tail derived from the injection of the transgene into fertilized oocytes, is analyzed.
- This DNA is digested with restriction enzymes, in particular PstI, and then the presence of the transgene is revealed by hybridization, for example under the conditions of stringent hybridization of this DNA with a labeled probe corresponding to a part of the transgene which is heterologous with respect to to the animal's genome, for example to the fragment of SV40 present only in the construct used to introduce the transgene, and for example the fragment
- the hybridization conditions are as follows: the nylon membranes are hybridized in a solution containing: 50% formamide, 5x SSC, 5x Denhardt's, 1% SDS, salmon sperm DNA 100 mg / ml, overnight at 42 ° C. Washes are carried out twice with 2x SSC, 0.1% SDS at
- the transgenic animal and a control animal are sacrificed and the RNA is prepared from different tissues.
- the RNA is analyzed by the RNA transfer technique (Northern blot) and the same probe always used as the probe defined above, and in particular the same fragment of SV40 as that which served to determine the presence of the transgene.
- a band corresponding to the expression of the transgene is found only in the pituitary gland of the transgenic mice and not in the control animal.
- the invention relates to the use of a non-human transgenic mammal animal, capable of overexpressed, particularly on lactotrophs in the pituitary, isoform D 2 L and / or D isoform S 2 of the dopamine D 2 receptor.
- the overexpression of DL compared to the normal expression can be approximately 7 times.
- the overexpression of D 2 S compared to the normal expression can be approximately 10 times.
- the overexpression is dependent on the promoter used, but also on the place of insertion of the transgene in the genome. This means that even using the same promoter, one is not sure of obtaining the same expression in two independent animals, because in one animal one can have integration of the transgene in a part of the genome more active compared to the other , or have multiple copies inserted in one case but not in the other. It is only a screening between several transgenic lines which makes it possible to possibly select two animals with similar levels of expression for the same gene or for two different genes having the same promoter.
- the overexpression of the D 2 L isoform or the overexpression of the DS isoform can be determined using the RNAse enzyme digestion protection technique, by comparing the intensity of the band corresponding to each isoform in a mouse. control and a transgenic mouse. The value of the intensity of the band obtained with RNA from control mice (since there is RNA normally present in the control animal) is subtracted from that obtained from transgenic mice.
- the invention relates to the use of a non-human transgenic mammalian animal which no longer expresses the DL isoform, or which no longer expresses either the DL isoform or the D 2 S isoform. .
- the absence of expression of the D 2 L isoform can be determined as follows.
- mice where expression of the DL form has been eliminated are first identified relative to the DNA by analysis with the DNA transfer method (Southern blot), using a probe consisting of a fragment. containing all or part of the D 2 receptor region which contains the insertion of 87 base pairs defined above, in particular a genomic probe shown in FIG. 4, EcoRI-BglI.
- RNA extracted from the striatum and the pituitary gland as well as other tissues is analyzed by the technique of protection against digestion by the enzyme RNAse.
- tissue that do not express the receptor and that serve as controls such as the cerebellum.
- the pharmacological properties are analyzed by binding of a specific ligand D 2 , for example in striatum or pituitary homogenates.
- the absence of expression of the D 2 L isoform and of D 2 S can be determined by one of the following methods: 1) Mice in which the expression of the form D 2 L and D 2 S has been eliminated , are verified by DNA transfer (Southern blot) with DNA extracted from the mouse tail cut with the Spel enzyme and hybridized with the PstI probe corresponding to the fragment indicated in FIG. 3.
- Hybridization of this probe clearly shows that the band corresponding to the wild type D receptor (that is to say normally present in animals) is no longer present in animals homozygous for the mutation.
- RNA transfer (Northern blot) of RNA extracted from tissues expressing D 2 , in particular striatum, shows that there is no longer any expression of RNA corresponding to the wild-type gene.
- the probe used in this case comprises the 5 ′ part of the cDNA of the D 2 gene, in particular a SmaI-PvuII fragment of 454 base pairs can be used (FIG. 9A).
- the invention relates to a non-human transgenic mammal animal or mammalian cells containing in their genome a construct chosen from:
- a promoter allowing the overexpression of the DS isoform on the lactotropic cells of the pituitary gland, in particular the prolactin promoter, + cDNA of D 2 S + a transcription terminator, in particular a part of the genome of the SV40 virus containing the site polyadenylation; - a promoter allowing the overexpression of the D 2 L isoform on the lactotropic cells of the pituitary gland, in particular the prolactin promoter, + D 2 L cDNA + a transcription terminator, in particular a part of the genome of the SV40 virus containing the polyadenylation site, and a promoter allowing the overexpression of the DS isoform on the lactotropic cells of the pituitary gland, in particular the prolactin promoter, + D 2 S cDNA + a transcription terminator, in particular a
- cDNA of DL or cDNA of DS also includes any nucleotide sequence derived from the sequences represented respectively in FIGS. 5 and 6, namely by addition, deletion or mutation of one or more bases, provided that the coded protein corresponding to this nucleotide sequence has biological properties equivalent to those of D receptors.
- DL or DS cDNA we also designate any nucleotide sequence capable of hybridizing respectively with the sequences represented in FIGS. 5 and 6, or taking into account the degeneration of the genetic code, capable of coding for the proteins respectively represented on Figures 5 and 6.
- Non-human transgenic mammalian animals are such that there is overexpression of DL and / or D 2 S on the lactotropic cells of the pituitary gland.
- the invention relates to a non-human transgenic mammal animal or mammalian cells containing the D receptor gene in which a fragment of the D 2 gene containing exon 2 or a fragment of the D gene containing exon 6 is replaced by all or part of a marker gene under the control of an appropriate promoter, in particular the neomycin resistance gene (neo) under the control of the phosphoglycerate kinase -1 promoter (PGK-1).
- an appropriate promoter in particular the neomycin resistance gene (neo) under the control of the phosphoglycerate kinase -1 promoter (PGK-1).
- the invention also relates to the transgenic constructs chosen from: a) a promoter allowing the overexpression of the D 2 L isoform on the lactotropic cells of the pituitary gland, in particular the prolactin promoter, + D 2 L cDNA + a terminator of transcription, in particular a part of the SV40 virus genome containing the polyadenylation site and in particular: BamHI-HindII fragment (-3000 / -l-33) of the prolactin promoter + Smal-EcoRV fragment of the D 2 cDNA L + BamHI-BglII fragment of SV40, b) a promoter allowing the overexpression of the isoform D 2 S on the lactotropic cells of the pituitary gland, in particular the prolactin promote
- the invention relates to cell cultures containing one of the above transgenic constructs.
- These cell cultures can be obtained either from cells taken from transgenic animals as defined above or either from cell lines using the above transgenic constructs using standard cell transfection techniques.
- the invention also relates to cell cultures, in which the cells are such that exon 2 or exon 6 of the D gene has been removed from one of the two alleles.
- the invention also relates to a non-human transgenic mammal as obtained by introduction, in particular microinjection into a fertilized oocyte, of one of the above transgenic constructions, insertion of the embryo into a surrogate mother and development of the embryo to term.
- the above non-human transgenic mammals contain one of the constructions a), b) or c), as defined above.
- the invention also relates to a non-human transgenic mammal as obtained by introduction into a blastocyte of embryonic stem cells (ES cells) comprising in their genome one of the above-mentioned transgenic constructions, obtained by homologous recombination, selection of male chimeric animals according to a criterion corresponding to the ES line, crossing of the selected animals with mice, in particular C57 Black 6 mice, to obtain animals heterozygous with respect to one of the above-mentioned constructions and possibly crossing of two heterozygotes to obtain an animal homozygous with in relation to one of the above constructions.
- ES cells embryonic stem cells
- the constructions referred to in the above transgenic animals are the constructions d) and e), as defined above.
- the invention also relates to a transgenic mammal as produced by crossing transgenic animals expressing one of the transgenic constructs defined above.
- the transgenic animals containing construct c) can be obtained either by introducing the constructs a) and b) into their genome, or by crossing a transgenic animal containing the construction a) with a transgenic animal containing the transgenic construction b).
- the invention also relates to a method for obtaining a transgenic model for the study of pathologies involving D 2 receptors, and to their treatment, comprising the introduction into the cells or embryos of non-human mammals of a construct chosen from:
- a promoter allowing the overexpression of the D 2 L isoform on the lactotropic cells of the pituitary gland, in particular the prolactin promoter, + D 2 L cDNA + a transcription terminator, in particular a part of the genome of the SV40 virus containing the polyadenylation site and in particular: BamHI-HindII fragment (-30O0 / + 33) of the prolactin promoter + Smal-EcoRV fragment of D 2 L cDNA + BamHI-BglII fragment of SV40,
- a promoter allowing the overexpression of the isoform D 2 S on the lactotropic cells of the pituitary gland, in particular the promoter of prolactin, + cDNA of D 2 S + a transcription terminator, in particular a part of the genome of the SV40 virus containing the polyadenylation site and in particular: BamHI-HindII fragment (-3000 / + 33) of the prolactin promoter + SmaI-EcoRV fragment of the D 2 S cDNA -I- BamHI-BglII fragment of SV40,
- D 2 L cDNA + a transcription terminator in particular a part of the SV40 virus genome containing the polyadenylation site and in particular: BamHI-HindII fragment (-3000 / + 33) of the prolactin promoter, + Smal fragment EcoRV of the D 2 L cDNA + BamHI-BglII fragment of SV40, - and a promoter allowing the overexpression of the D 2 S isoform on the lactotropic cells of the pituitary gland, in particular the prolactin promoter, + cDNA of D 2 S + a terminatc.
- BamHI-HindII fragment (-3000 / + 33) of the prolactin promoter 4- Smal-EcoRV fragment of the D 2 S cDNA + BamHI-BglII fragment of SV40,
- the gene for D 2 in which a fragment containing exon 2 is replaced by a marker gene under the control of an appropriate promoter in particular a cassette containing the neo gene under the control of the PGKI promoter, and in particular the gene for D 2 in which the 0.9 Kb Ncol genomic fragment containing exon 2 is replaced by the PGK-neo cassette.
- the D 2 gene in which a fragment containing exon 6 is replaced by a marker gene under the control of an appropriate promoter in particular by a cassette containing the neo gene under the control of the PGKI promoter, and in particular the D 2 gene in which the BglII-BglII genomic fragment containing exon 6 is replaced by the PGK-neo cassette.
- the invention also relates to a method for screening for drugs active on pathologies involving D 2 receptors, in particular prolactinomas, or certain neurodegenerative pathologies such as Parkinson's disease, Huntington's disease or schizophrenia, comprising:
- a promoter allowing the overexpression of the D 2 L isoform on the lactotropic cells of the pituitary gland, in particular the prolactin promoter, + D 2 L cDNA + a transcription terminator, in particular a part of the genome of the SV40 virus containing the polyadenylation site and in particular: BamHI-HindII fragment (-3000 / + 33) of the prolactin promoter + Smal-EcoRV fragment of the D 2 L cDNA + BamHI-BglII fragment of SV40,
- a promoter allowing the overexpression of the isoform D 2 S on the lactotropic cells of the pituitary gland, in particular the promoter of prolactin, -I- cDNA of D 2 S + a transcription terminator, in particular a part of the genome of the virus SV40 containing the polyadenylation site and in particular: BamHI-HindII fragment (-30O0 / + 33) of the prolactin promoter + Smal-EcoRV fragment of D 2 S cDNA + BamHI-BglII fragment of SV40,
- a promoter allowing the overexpression of the DL isoform on the lactotropic cells of the pituitary gland, in particular the prolactin promoter, + cDNA of D 2 L + a transcription terminator, in particular a part of the genome of the SV40 virus containing the site polyadenylation and in particular: BamHI-HindII fragment (-3000 / + 33) of the prolactin promoter + SmaI-EcoRV fragment of DL cDNA + BamHI-BglII fragment of SV40,
- DS cDNA + a transcription terminator in particular a part of the SV40 virus genome containing the polyadenylation site and in particular: BamHI-HindII fragment (-3000 / + 33) of the prolactin promoter + Smal-EcoRV fragment of the CDNA of D 2 S + BamHI-BglII fragment of SV40, - the D 2 gene in which a fragment containing exon 2 is replaced by a marker gene under the control of an appropriate promoter, in particular a cassette containing the gene neo under the control of the promoter PGKI, and in particular the D 2 gene in which the genomic fragment of 0.9 Kb Ncol, containing exon 2 is replaced by the PGK-neo cassette,
- Prolactinomas are very common tumors in women. The origins of these tumors are not yet known, but a malfunction of dopaminergic control at the lactotropic level is implicated.
- the transgenic animals of the invention may serve as a model for the study and the determination of the treatment of such a pathology. Indeed, in these animals, the normal balance between the expression of the form D 2 L and D 2 S is altered, which makes it possible to study the phenotype corresponding to such an alteration.
- dopamine D 2 agonists and antagonists can be tested for their effectiveness in increasing or suppressing the phenotypes obtained.
- the overexpression of the D 2 S form causes a significant decrease in the level of prolactin with the consequence of a lactation deficit in females.
- Remoxipride is an antagonist known to have a high specificity for binding with D 2 S, which makes it possible to test whether the injection of this substance makes it possible to eliminate the deficit in the level of prolactin in transgenic animals overexpressing D 2 S.
- D 2 receptors are involved in the control of several physiological functions such as movement coordination, vision, regulation of the synthesis of certain hormones, etc. We can therefore highlight the deficits linked to the absence of these receptors. For example, one of the phenotypes linked to the absence of the D 2 receptor can be poor coordination of movements. These animals can then be used to test compounds acting on the stimulation of movement, in order to see if this deficit can be compensated.
- the transgenic animals of the invention are advantageous for screening the substances currently used with specificity D in order to define if this is really the case.
- the transgenic animals of the invention therefore constitute a remarkable tool in this sense.
- transgenic animals of the invention expressing only the DS form of the receptor can be used to develop specific D 2 S antagonists, with the possibility of being able to test these antagonists in vivo in the absence of the D 2 L receptor. .
- the invention relates to a method for screening for drugs active in an animal model in which the prolactin levels are reduced compared to the normal state using transgenic animals overexpressing D 2 S, comprising: * administration to a transgenic non-human mammal or to transgenic non-human mammal cells containing a construct chosen from:
- a promoter allowing the overexpression of the isoform D 2 S on the lactotropic cells of the pituitary gland, in particular the promoter of prolactin, + cDNA of D 2 S + a transcription terminator, in particular a part of the genome of the SV40 virus containing the polyadenylation site and in particular: BamHI-HindII fragment (-3000 / + 33) of the prolactin promoter + Smal-EcoRV fragment of the D 2 S cDNA + BamHI-BglII fragment of SV40, of the compound to be tested, * and the determination of the increase in prolactin level.
- the invention relates to a method for screening for drugs active on prolactinomas using transgenic animals overexpressing D L, comprising:
- a promoter allowing the overexpression of the D 2 L isoform on the lactotropic cells of the pituitary gland, in particular the prolactin promoter, + D 2 L cDNA + a transcription terminator, in particular a part of the SV40 virus genome containing the polyadenylation site and in particular: BamHI-HindII fragment (-3000 / + 33) of the prolactin promoter + Smal-EcoRV fragment cDNA of D 2 L + fragment BamHI-BglII of SV40, of the compound to be tested,
- the invention relates to a method for screening for drugs which are active on neurodegenerative diseases such as Parkinson's disease, Huntington's disease or schizophrenia using transgenic animals in which D 2 S and DL are no longer expressed, including
- the gene for D 2 in which a fragment containing exon 2 is replaced by a marker gene under the control of an appropriate promoter in particular a cassette containing the neo gene under the control of the PGKI promoter, and in particular the gene for D 2 in which the 0.9 Kb Ncol genomic fragment containing exon 2 is replaced by the PGK-neo cassette of the compound to be tested, * and the determination of the improvement of the neurodegenerative state.
- mice can represent a study model for diseases such as Parkinson's disease or schizophrenia.
- drugs which could be tested in the transgenic animals of the invention can also improve diseases which do not have abnormalities of the D 2 receptor at their base.
- a transgene is defined as a fragment of DNA which is inserted artificially (i.e. other than by sexual reproduction) in a cell and which becomes part of the genome of the animal which develops from that cell, or that is modified from the DNA normally present in the cell.
- Such a transgene may be a gene homologous to a natural gene of the transgenic animal but which is inserted into the genome of the animal at a position which differs from that of the natural counterpart, or else such a transgene may correspond to the homologous gene. modified, in particular by deletion of a DNA fragment of said natural gene.
- the invention also relates to a process for the preparation of a transgenic animal, carrying the aforementioned transgene, which can be raised stably to give offspring comprising cells which contain the gene stably.
- This process can include: - the removal of a fertilized oocyte from a first female animal,
- said female animal begins gestation with respect to the embryo derived from the fertilized oocyte containing the transgene
- the embryo develops into a transgenic animal
- the transgenic animal is born from the second female animal.
- FIG. 1C shows the BamHI-BglII fragment which serves as a probe to determine whether there has been integration of the transgene.
- PRLD 2 L the transgenic construction or the gene containing said construction, allowing the overexpression of DL
- PRLD S the transgenic construction or the gene containing said construct, allowing the overexpression of D 2 S.
- PRLD L or PRLD 2 S transgenic mice denotes mice containing the transgenic construct PRLD 2 L or PRLD 2 S.
- FIG. 2 represents the PCR analysis of mice comprising both the PRLD 2 L and PRLD 2 S construct.
- PCR polymerase chain reaction
- FIG. 3 shows the homologous recombination leading to the preparation of transgenic mice no longer expressing either D 2 S or D 2 L.
- FIG. 4 shows the homologous recombination leading to the preparation of transgenic mice no longer expressing D 2 L.
- FIG. 6 represents the amino acid and nucleotide sequence of
- FIG. 7 represents the analysis of the RNA originating from the pituitaries of transgenic mice PRLD 2 L and PRLD 2 S compared to wild mice, and shows the overexpression of the isoforms D 2 L or D 2 S.
- RNAse from the pituitaries of transgenic mice PRLD 2 L, PRLD 2 S and their controls (C) (non-transgenic sibling, of the same litter), from the hybrid RNA probe to the RNA corresponding to the messenger RNA of the D 2 gene, this probe being described in the text (AccI fragment in pBluescript, plasmid linearized by Bsu36I).
- C non-transgenic sibling, of the same litter
- the arrows indicate the bands corresponding to the expected size for D 2 L (202 nucleotides) and D 2 S (130 nucleotides).
- FIG. 8 represents the protection with the RNAse enzyme of RNA originating from tissues of mice (normal or wild mice, heterozygous or homozygous) in which the D 2 L form of the receptor has been eliminated.
- RNAs from striatum, substantia nigra, cortex and cerebellum of normal (+ / +), heterozygous (+/-) and homozygous (- / -) mice were hybridized with the probe corresponding to the plasmid containing the AccI fragment cut by Bsu36I and transcribed by the T3 enzyme. The disappearance of the band corresponding to the D 2 L form is clearly observed in homozygous mice. In this photograph, bands corresponding to the D 2 transcript are observed only in the striatum, and the substantia nigra as expected, the other regions of the brain used (cortex, cerebellum) serving as negative control.
- FIG. 9 represents the absence of expression of D 2 in the transgenic mice obtained by homologous recombination.
- D 2 receptor For the analysis of the D 2 receptor, 15 ⁇ g of membranes are incubated in a 50 mM Tris-HCl solution, pH 7.7, containing 120 mM NaCl, 5 mM KC1, 2 mM CaCl 2 , 1 mM MgCl 2 , 0 , 1% ascorbic acid and 3H-spiperone (10-600 pM; specific activity 114 Ci / mmol; Amersham) at 37 ° C for 60 min. Nonspecific binding is determined in the presence of 1 ⁇ M (+) - butaclamol (RBI, Natick, MA).
- the radioactive ligand bound to the membranes is recovered by rapid filtration through Whatmann GF / B glass fiber filters.
- the filters are washed three times with 5 ml of 50 mM Tris-HCl, pH 7.7 cold.
- the data is analyzed with the LIGAND program (BIOSOFT). On the abscissa, the amount of 3H-spiperone linked to the receptor is indicated.
- the prolactin gene promoter was used.
- mice born from these injections are tested for the presence of the transgenic construct by analyzing genomic DNA obtained from a small sample of the tail.
- the DNA obtained is tested by DNA transfer analysis (Southern blot) digested with PstI, brought into contact with a probe consisting of a fragment of SV40 BamHI-BglII labeled with 32p ; only transgenic mice are revealed in this way, since the DNA of wild (i.e. non-transgenic) mice does not hybridize with this probe.
- Mouse lines are obtained by crossing male or female transgenic mice with C57 Black 6 mice from Jackson
- mice are tested for transgene expression by analysis of pituitary mRNA by the RNAse protection method, using a specific probe (AccI fragment in pBluescript linearized by Bsu36I) of the D 2 receptor and by transfer analysis RNA (Northern blot), using the SV40 BamHI-BglII fragment labeled with 32p.
- mice overexpressing both isoforms of D 2 receptors are crossed female LTIP 2 L heterozygous males with LTIP 2 S.
- an amplification technique is used polymerase chain reaction (PCR).
- the genomic DNA of the mouse tail is amplified using synthetic oligonucleotides belonging to the added fragment SV40 and for example extending from the nucleotide at position 4457 to the nucleotide at position 4477 of the genome of the SV40 virus, and to the cDNA of the receptors dopamine.
- Oligonucleotide primers are chosen from the D receptor cDNA region upstream of the present insertion into D 2 L (for example from position 830 to position 850 of mouse cDNA). Two fragments of different length are obtained, highlighting the doubly transgenic character (see FIG. 2).
- Example 3 Preparation of transgenic mice no longer expressing DS or DL (homologous recombination).
- cloned genomic DNA corresponding to the D 2 receptor site is isolated from a genomic library of ES / EMBL3 mice: (Sambrook J., Fritsch, EF, Maniatis, T., Molecular Cloning, 2nd edition, Ed. Cold Spring Harbor Laboratory Press, 1989, vol. 2, chapter 9).
- the ES cells come from the mouse strain 129 of agouti color (Jackson Laboratory).
- a 6.5 kb Kpnl-Sall mouse genomic fragment containing exon 2 (Karen L.
- DMEM Dulbecco modified Eagle's medium
- FCS fetal calf serum
- the cells are then transferred to a non-selective medium (Dulbecco) for 2 days and selected in 150 ⁇ g / ml of gentamycin (G418) for 10 days.
- the resistant ES clones are isolated and placed in microwells containing a cellular nourishing layer (irradiated fibroblasts) and then multiplied for DNA analysis by the DNA transfer technique (Southern blot).
- ES cells carrying the D gene generated by homologous recombination are injected into blastocyte C57BL / 6 embryos from Jackson Laboratory using standard techniques (Bradley, A. (1987) Production and analysis of chimaeric mice. In teratocarcinomas and Embryonic Stem Cells: A practical Approach, Robertson, EJ, ed.
- the chimeric descendants are identified by the color of the hair corresponding to the chimeric phenotype (gray and black color) and are crossed with C57BL / 6 mice.
- the transmission of the germ line of the mutant allele is determined by DNA transfer (Southern blot) from the DNA taken from the tail isolated from the "agouti" progeny by digestion with Spel. Mice homozygous towards the D mutation are obtained by crossing mutant heterozygous mice.
- Example 4 Preparation of mice no longer expressing D L by homologous recombination.
- a 4.8 Kb EcoRI genomic fragment containing exon 3 and exon 4 is subcloned into pBluescript, a 1.5 Kb BglII fragment surrounding l exon 6 is deleted and replaced by the PGK-neo cassette gene.
- a 1.5 Kb BglII fragment surrounding l exon 6 is deleted and replaced by the PGK-neo cassette gene.
- another 5.8 Kb EcoRI genomic fragment going from exon 5 to exon 7 is fused to provide a fragment of greater homology.
- the viral gene for simple herpes thymidine kinase (TK) under the control of the PGK promoter is included at the 3 'end of the vector.
- the targeting vector is linearized at the unique NotI site in the multiple cloning site before electroporation of ES D3 cells (Dr. Kemler Institut Max Planck, Friborg,
- Electroporated ES D3 cells are selected from 150 ⁇ g / ml gentamycin (G418) + 2 ⁇ M gancyclovir for 10 days.
- the targeted clones are identified by transfer of genomic DNA (Southern blot) cut with Bgl1 and hybridized to an external probe originating from a fragment of D 2 surrounding exon 8 and digested with EcoRI-BglI (FIG. 4). Digestion of DNA with Bgl1 gave a fragment of 6.8 Kb from wild type and 4.5 Kb from targeted allele.
- the process for generating mutant mice no longer having the D 2 L isoform is carried out according to what has been described from the mice no longer expressing either the D 2 S receptor or the D 2 L receptor.
- Example 5 Characterization of the mice no longer expressing the D receptor (D 2 knockout mice) or no longer expressing D 2 L.
- D2 knockout mice exhibit a Parkinsonian phenotype, very reduced movement, poor coordination and akinesia.
- the locomotion phenotype of these mice demonstrates that the D 2 receptor, and not the other dopaminergic receptors (such as Di, D3, D4 and D5), is the dopaminergic receptor responsible for this function.
- the similarity of the phenotype between D - / - mice (expressing only the D 2 receptor) and Parkinson's disease suggests that D 2 receptors are the most affected of dopaminergic receptors by the lack of dopamine in patients. This observation validates these mice as a model for studying Parkinson's disease. We can study what other neural circuits are affected by the lack of D 2 receptor activity and try drugs capable of reducing these effects on these animals.
- Parkinson's disease is now treated only by taking L-DOPA, the metabolic precursor of dopamine. It has also been found that in the animals of the invention, there is an increase in the synthesis of the enzyme G AD (glutamic acid decarboxylase) determined at the level of the mRNA by in situ hybridization and therefore may be of the neurotransmitter GABA ( ⁇ -aminobutyric acid).
- G AD glutamic acid decarboxylase
- mice of the invention can serve as a tool for validating compounds targeting other dopaminergic receptors of the D 2 subfamily, such as D3 and D4, or of the Di subfamily, such as O and D5 with specificity. for D 2 only and not for D ⁇ and D 2 .
- D 2 - / - mice develop hypertrophies of the intermediate and anterior lobes of the pituitary gland, these phenomena being accompanied by an increased synthesis of the hormones resulting from the transcription of the propiomelanocortin gene, and of prolactin.
- These D 2 - / - mice, and especially the females, also develop prolactinoma-like tumors in a later phase of their life (after 8 months of birth).
- a very detailed analysis of these phenomena allows us to know what are the causes of this disorderly growth of pituitary cells and to find treatments capable of preventing them.
- the dopaminergic receptor being very widespread at the central, pituitary, but also at the level of the olfactory system, in the retina, etc.
- the invention according to an advantageous embodiment, relates to a model, for the study of human pathologies due to a lack or reduction of the D receptor.
- mice which no longer have the long form of the D 2 receptor the D 2 L - / -, they clearly have a phenotype different from the D - / -.
- the phenotype consists of hyperlocomotion compared to wild mice.
- These mice will make it possible to assign the function of each isoform of the D 2 receptor in vivo.
- the D 2 S form can represent the dopaminergic auto-receptor, that is to say the receptor which controls the synthesis of dopamine
- the industrial interest of the animals of the invention consists in their use for testing compounds which do not act on the D 2 O and D 2 receptor for adjusting the synthesis of dopamine.
- the invention also relates to animals which are generated by crossing D 2 - / - and DL - / - mice with other mutant animals, vis-à-vis other receptor genes or molecules involved in the pathway. dopaminergic metabolic.
- the invention also relates to the use of the mice of the invention for testing vectors used for gene therapy of diseases such as Parkinson's disease, schizophrenia and other neurodegenerative diseases.
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Abstract
The use of a non human transgenic mammalian animal in which the expression of the gene coding for the dopamine D2 receptor has been modified in relation to the normal expression, particularly on the lactotropic cells of the pituitary gland, for screening for a drug active against pituitary gland diseases involving the dopamine D2 receptor is described.
Description
ANIMAL TRANSGENIQUE NON HUMAIN DANS LEQUEL L'EXPRESSION DU RECEPTEUR D2 EST MODIFIEE.NON-HUMAN TRANSGENIC ANIMAL IN WHICH THE EXPRESSION OF THE D 2 RECEPTOR IS MODIFIED.
L'invention a pour objet un animal transgénique on humain dans lequel l'expression du gène codant pour le récepteur D2 est modifiée.The subject of the invention is a transgenic or human animal in which the expression of the gene coding for the D 2 receptor is modified.
La dopamine est un neuromédiateur qui assure la régulation de beaucoup de fonctions physiologiques. Dans le système nerveux central, la dopamine est impliquée dans la coordination des mouvements, la perception visuelle, le savoir, l'émotion et dans le contrôle neuroendocrinien de la glande pituitaireDopamine is a neurotransmitter that regulates many physiological functions. In the central nervous system, dopamine is involved in the coordination of movement, visual perception, knowledge, emotion and in neuroendocrine control of the pituitary gland
(hypophyse). L'activation par la dopamine des récepteurs de membrane génère la formation de messagers secondaires intracellulaires. Les récepteurs de la dopamine appartiennent à la famille des récepteurs à sept domaines transmembranaires couplés aux protéines G du domaine 7TM (J. A. Gingrich et M. G. Caron, Annu. Rev. Neurosci., 16: 299 (1993)). Des études pharmacologiques et biochimiques ont décrit l'existence de deux classes de récepteurs dopaminergiques Dj et D2. Le récepteur Dj a été identifié comme activateur de l'adénylate cyclase (AC), au contraire de D2 qui est décrit comme inhibiteur du même effecteur cellulaire. Ces deux types de récepteurs diff rent dans leur affinité pharmacologique pour les antagonistes du récepteur de la dopamine classique tels que SCH23390 pour Dj et spipérone pour D2. Le clonage moléculaire de l'ADNc du récepteur D2 (J. R. Bunzow et al. , Nature, 336: 783 (1988)) a permis des criblages dans des conditions d'hybridation non stringentes d'ADNc et de banques génomiques avec l'isolement de 4 récepteurs dopaminergiques supplémentaires. Deux d'entre- eux, D3 (P. Sokoloff et al., Nature, 347: 146 (1990)) et D4 (H. H. M. Van Toi et al., Nature, 350: 610 (1991)) montrent des caractéristiques similaires au récepteur D2, c'est-à-dire une affinité picomolaire à nanomolaire pour la spipérone. Les deux autres dénommés Dj (A. Dearry et al., Nature, 347: 72 (1990)) et D5 (D. K. Grandy et al. , Proc. Natl. Acad. Sci. USA, 88: 9175(pituitary gland). Dopamine activation of membrane receptors generates the formation of intracellular secondary messengers. Dopamine receptors belong to the family of receptors with seven transmembrane domains coupled to G proteins of the 7TM domain (JA Gingrich and MG Caron, Annu. Rev. Neurosci., 16: 299 (1993)). Pharmacological and biochemical studies have described the existence of two classes of dopamine receptors Dj and D 2 . The Dj receptor has been identified as an activator of adenylate cyclase (AC), unlike D 2 which is described as an inhibitor of the same cellular effector. These two types of receptors differ in their pharmacological affinity for classical dopamine receptor antagonists such as SCH23390 for Dj and spiperone for D 2 . Molecular cloning of the D 2 receptor cDNA (JR Bunzow et al., Nature, 336: 783 (1988)) allowed screening under non-stringent hybridization conditions of cDNA and genomic libraries with isolation. of 4 additional dopaminergic receptors. Two of them, D3 (P. Sokoloff et al., Nature, 347: 146 (1990)) and D 4 (HHM Van Toi et al., Nature, 350: 610 (1991)) show characteristics similar to D 2 receptor, i.e. a picomolar to nanomolar affinity for spiperone. The other two called Dj (A. Dearry et al., Nature, 347: 72 (1990)) and D5 (DK Grandy et al., Proc. Natl. Acad. Sci. USA, 88: 9175
(1991)) présentent des caractéristiques pharmacologiques typiques des récepteurs Dj classiquement décrits. Les ADNc des récepteurs clones ont été exprimés dans des lignées cellulaires et leur profil pharmacologique a été défini ainsi que leurs propriétés à transduire le signal à l'intérieur de la cellule. Comme prévu, les récepteurs Dj et D5 sont des stimulateurs de l'adénylate cyclase. Le récepteur D5 a une affinité supérieure pour la dopamine à D1 . Le schéma est moins clair pour le groupe des récepteurs du type de D . A présent, le mécanisme par lequel les récepteurs D3 et D4 transduisent le
signal, reste incertain, bien qu'ils présentent un profil pharmacologique proche de D . Un rôle pour D3 comme autorécepteur a été proposé sur la base d'une affinité supérieure pour la dopamine, en comparaison de D2 (J. A. Gingrich et M. G. Caron, Annu. Rev. Neurosci., 16: 299 (1993)). Le récepteur D4 a une affinité supérieure (environ dix fois) à D2 et D3 pour la clozapine, un antipsychotique, à des concentrations qui sont actives in vivo, et il a été proposé comme cible de ce médicament. L'absence d'identification d'une voie de transduction pour ces récepteurs pourrait être due à l'absence de protéines G spécifiques dans les lignées cellulaires testées. La sous-famille entière des récepteurs à la dopamine est largement exprimé dans le système nerveux central, en particulier dans le caudate- putamen, le nucleus accumbens, l'hypothalamus et les tubercules olfactives. Les caractéristiques pharmacologiques communes entre les groupes D^ et D des récepteurs dopaminergiques sont également reflétées au niveau amino acide où une homologie de plus de 40% est observée sur la séquence totale entre les membres de chaque groupe.(1991)) have pharmacological characteristics typical of the classically described D j receptors. The cloned receptor cDNAs were expressed in cell lines and their pharmacological profile was defined as well as their properties to transduce the signal inside the cell. As expected, the Dj and D5 receptors are stimulators of adenylate cyclase. The D5 receptor has a higher affinity for dopamine than D1. The pattern is less clear for the group of D-type receptors. Now the mechanism by which the D3 and D4 receptors transduce the signal, remains uncertain, although they have a pharmacological profile close to D. A role for D3 as an autoreceptor has been proposed on the basis of a higher affinity for dopamine, in comparison with D 2 (JA Gingrich and MG Caron, Annu. Rev. Neurosci., 16: 299 (1993)). The D4 receptor has a greater affinity (about ten times) than D 2 and D3 for clozapine, an antipsychotic, at concentrations that are active in vivo, and it has been proposed as a target for this drug. The absence of identification of a transduction pathway for these receptors could be due to the absence of specific G proteins in the cell lines tested. The entire dopamine receptor subfamily is widely expressed in the central nervous system, particularly in the caudate-putamen, nucleus accumbens, hypothalamus and olfactory tubers. The pharmacological characteristics common between the D ^ and D groups of dopaminergic receptors are also reflected at the amino acid level where a homology of more than 40% is observed on the total sequence between the members of each group.
L'ADNc du premier récepteur D2 isolé code pour une protéine de 415 amino acides et a été isolé à partir d'une banque d'ADNc de cerveau de rat (J. R. Bunzow et al., Nature, 336: 783 (1988)). Le profil pharmacologique obtenu à partir de cellules transfectées l'identifie clairement comme un récepteur dopaminergique D2. Peu de temps après, un autre ADNc a été isolé à partir de sources différentes qui est 100% homologue au premier, à l'exception d'une insertion de 29 amino acides dans le troisième domaine intracellulaire du récepteur. Cet ADNc code une protéine de 444 amino acides, que l'on a trouvé chez le rat, la souris, les bovins et l'humain (l'ADNc humain a 445 amino acides). (J. A. Gingrich et M. G. Caron, Annu. Rev. Neurosci., 16: 299 (1993); J.-P. Montmayeur et al., Febs lett., 278: 239 (1991)). Les analyses d'ADN génomique ont démontré que les deux ADNc sont des produits du même gène générés par un mécanisme d'épissage alternatif qui ajoute un exon de 87 paires de base dans le plus grand des ARN messagers. Ces récepteurs ont été désignés par D S pour la forme la plus courte, et par D2L pour la forme la plus longue.The first isolated D 2 receptor cDNA codes for a protein of 415 amino acids and was isolated from a rat brain cDNA library (JR Bunzow et al., Nature, 336: 783 (1988)) . The pharmacological profile obtained from transfected cells clearly identifies it as a dopamine D 2 receptor. Shortly thereafter, another cDNA was isolated from different sources which is 100% homologous to the first, except for insertion of 29 amino acids into the third intracellular domain of the receptor. This cDNA codes for a protein of 444 amino acids, which has been found in rats, mice, cattle, and humans (human cDNA has 445 amino acids). (JA Gingrich and MG Caron, Annu. Rev. Neurosci., 16: 299 (1993); J.-P. Montmayeur et al., Febs lett., 278: 239 (1991)). Genomic DNA analyzes have shown that the two cDNAs are products of the same gene generated by an alternative splicing mechanism that adds an 87 base pair exon to the larger of the messenger RNAs. These receptors have been designated by DS for the shortest form, and by D 2 L for the longest form.
La structure du fragment génomique contenant le gène D2 a été déterminée. On a trouvé 8 exons dans toutes les espèces analysées, avec une caractéristique surprenante représentée par la présence d'un très grand intron situé en amont du premier exon codant. La présence d'une aussi grande séquence intronique correspond à un mécanisme de contrôle de l'expression du gène D . Le clonage de la région du promoteur du gène D a été rapporté;
étant donné qu'aucun trait caractéristique n'a été décrit, il semble correspondre aux caractéristiques d'un promoteur ubiquitaire. De façon intéressante, les deux isoformes du récepteur D2 sont co-exprimés dans tous les tissus testés avec un rapport qui favorisent normalement l'expression de D2L. (J.-P. Montmayeur et al., Febs lett., 278: 239 (1991)). S'agissant de la pharmacologie, ces deux isoformes ont des profils similaires et se lient à l'antagoniste spipérone avec une affinité semblable. L'activation de ces deux récepteurs D2 résulte principalement dans l'inhibition de l'adénylate cyclase. Cependant, il apparaît qu'ils sont également capables d'activer d'autres voies de transduction de signal. (J. A. Gingrich et M. G. Caron, Annu. Rev.The structure of the genomic fragment containing the D 2 gene has been determined. Eight exons were found in all the species analyzed, with a surprising characteristic represented by the presence of a very large intron located upstream of the first coding exon. The presence of such a large intronic sequence corresponds to a mechanism for controlling the expression of the D gene. Cloning of the D gene promoter region has been reported; since no characteristic trait has been described, it seems to correspond to the characteristics of a ubiquitous promoter. Interestingly, the two isoforms of the D 2 receptor are co-expressed in all of the tissues tested with a ratio which normally favor the expression of D 2 L. (J.-P. Montmayeur et al., Febs lett., 278 : 239 (1991)). With regard to pharmacology, these two isoforms have similar profiles and bind to the spiperone antagonist with similar affinity. The activation of these two D 2 receptors mainly results in the inhibition of adenylate cyclase. However, it appears that they are also capable of activating other signal transduction pathways. (JA Gingrich and MG Caron, Annu. Rev.
Neurosci. , 16: 299 (1993)). Ces observations, ainsi que le fait que les deux récepteurs diffèrent seulement d'une insertion dans la troisième boucle intracellulaire, suggèrent que les deux récepteurs peuvent différer dans leur capacité à se coupler à une ou plusieurs protéines G. L'un des aspects de l'invention est de fournir des animaux mammifères transgéniques non humains susceptibles de surexprimer l'un et/ou l'autre isoforme du récepteur D2.Neurosci. , 16: 299 (1993)). These observations, as well as the fact that the two receptors differ only from insertion into the third intracellular loop, suggest that the two receptors may differ in their ability to couple to one or more G proteins. One aspect of the The invention is to provide non-human transgenic mammalian animals capable of overexpressing one and / or the other isoform of the D 2 receptor.
L'un des autres aspects de l'invention est de fournir des animaux mammifères transgéniques non humains dans lesquels soit l' isoforme D2L n'est plus exprimé, soit les deux isoformes D2L et D2S ne sont plus exprimés.One of the other aspects of the invention is to provide non-human transgenic mammalian animals in which either the D 2 L isoform is no longer expressed, or the two D 2 L and D 2 S isoforms are no longer expressed.
L'un des autres aspects de l'invention est de fournir un modèle animal susceptible de cribler des médicaments actifs sur des pathologies impliquant les récepteurs D2.One of the other aspects of the invention is to provide an animal model capable of screening drugs which are active on pathologies involving the D 2 receptors.
L'invention a pour objet l'utilisation d'un animal mammifère transgénique non humain dont l'expression du gène codant pour le récepteur dopaminergique D2 est modifiée, notamment sur les cellules lactotropes de l'hypophyse, par rapport à une expression normale, notamment sur les cellules lactotropes de l'hypophyse, pour la détermination d'un médicament actif sur les pathologies impliquant le récepteur D2. Le terme "mammifère" inclut tous les mammifères à l'exception des humains, avantageusement les rongeurs et notamment les souris.The subject of the invention is the use of a non-human transgenic mammalian animal whose expression of the gene coding for the dopaminergic receptor D 2 is modified, in particular on the lactotropic cells of the pituitary gland, compared to a normal expression, in particular on the lactotropic cells of the pituitary gland, for the determination of a drug which is active on pathologies involving the D 2 receptor. The term "mammal" includes all mammals except humans, advantageously rodents and in particular mice.
L'expression normale du récepteur dopaminergique D2 peut être définie par plusieurs méthodes:The normal expression of the dopaminergic receptor D 2 can be defined by several methods:
1) Détermination de l'ARNm correspondant au gène D2: ceci est possible par la technique de transfert d'ARN (Northern blot) dans laquelle les1) Determination of the mRNA corresponding to the D 2 gene: this is possible by the RNA transfer technique (Northern blot) in which the
ARNm sont séparés sur gel i'agarose dénaturant par électrophorèse, puis les ARN sont transférés et fixés sur une membrane de type nitrocellulose ou nylon. Pour révéler la présence des ARN correspondants au gène D , on
utilise une sonde correspondant à la totalité de l'ADNc du gène. (On peut utiliser indifféremment l'ADNc de D2L ou de D2S).MRNAs are separated on a denaturing agarose gel by electrophoresis, then the RNAs are transferred and fixed on a nitrocellulose or nylon type membrane. To reveal the presence of RNA corresponding to the D gene, we uses a probe corresponding to the entire cDNA of the gene. (The D 2 L or D 2 S cDNA can be used interchangeably).
2) Détermination de la quantité de protéine correspondant au récepteur D : ceci est possible en étudiant la liaison d'un ligand spécifique vis à vis de D (agoniste ou antagoniste) tel que spipérone marquée, aux récepteurs présents dans un homogénat de tissu (striatum ou hypophyse). En particulier, la constante de dissociation Kd de plusieurs ligands D spécifiques est connue. On sait, par exemple, que le Kd pour le ligand 3H-spipérone est dans les valeurs picomolaires (pM). On sait donc qu'une courbe de saturation avec ce ligand sur des extraits de membranes contenant les récepteurs D de striatum, d'hypophyse ou de substantia nigra et l'analyse par la méthode de Scatchard (détermination du nombre de sites du récepteur) des résultats obtenus à partir des courbes de saturation, doit donner des valeurs d'affinité (Kd) pour la spipérone de l'ordre du picomolaire, pour que l'on puisse affirmer qu'il s'agit d'une liaison D2.2) Determination of the amount of protein corresponding to the D receptor: this is possible by studying the binding of a specific ligand with respect to D (agonist or antagonist) such as labeled spiperone, to the receptors present in a tissue homogenate (striatum or pituitary gland). In particular, the dissociation constant Kd of several specific D ligands is known. We know, for example, that the Kd for the ligand 3H-spiperone is in the picomolar values (pM). We therefore know that a saturation curve with this ligand on extracts of membranes containing the D receptors of striatum, pituitary or substantia nigra and the analysis by the Scatchard method (determination of the number of receptor sites) of results obtained from the saturation curves, must give affinity values (Kd) for spiperone of the order of a picomolar, so that it can be affirmed that it is a D 2 bond.
Ceci permet donc de quantifier la quantité correspondant au récepteur D dans l'extrait.This therefore makes it possible to quantify the quantity corresponding to the D receptor in the extract.
Par ailleurs, pour discriminer entre la forme D S et D L du récepteur D , on utilise la méthode de protection à l'enzyme RNAse. Pour cela, on a clone le fragment AccI de 370 paires de bases (positions de sites AccI: 700 etIn addition, to discriminate between the D S and D L forms of the D receptor, the RNAse enzyme protection method is used. For this, the 370 base pair AccI fragment was cloned (positions of AccI sites: 700 and
1070 de l'ADNc de D2 de souris), à partir de l'ADNc du récepteur D L dans le vecteur plasmidique pBluescript dans le site AccI. Pour la préparation de la sonde, ce plasmide est ensuite linéarisé par coupure à l'enzyme Bsu36I et transcrit in vitro à l'aide de la polymérase T3 en utilisant des nucléotides marqués au 32ps p0Ur avoir une sonde à ARN marquée. La taille de cette sonde est de 300 nucléotides. Les fragments protégés sont de 202 nucléotides pour D L et de 130 nucléotides pour D2S. (Voir figures 7 ou 8).1070 mouse D 2 cDNA), from the DL receptor cDNA in the plasmid vector pBluescript in the AccI site. For the preparation of the probe, this plasmid is then linearized by cleavage with the enzyme Bsu36I and transcribed in vitro using the T3 polymerase using nucleotides labeled with 32 p s or having a labeled RNA probe. The size of this probe is 300 nucleotides. The protected fragments are 202 nucleotides for DL and 130 nucleotides for D 2 S. (See FIGS. 7 or 8).
Cette sonde est utilisée pour l'hybridation avec les ARN provenant des régions exprimant le récepteur D (striatum, hypophyse). Les hybrides sont ensuite digérés avec l'enzyme RNAse et on obtient deux fragments: l'un correspondant à D2L de 202 paires de bases, et l'autre correspondant à D S de 130 paires de base. Cette analyse est dite quantitative car elle permet d'évaluer exactement la proportion des ARN correspondant à l'un ou l'autre des isoformes. Pour déterminer s'il y a modification de l'expression du gène D , on peut procéder comme indiqué ci-après.This probe is used for hybridization with RNA from regions expressing the D receptor (striatum, pituitary gland). The hybrids are then digested with the RNAse enzyme and two fragments are obtained: one corresponding to D 2 L of 202 base pairs, and the other corresponding to DS of 130 base pairs. This analysis is called quantitative because it makes it possible to evaluate exactly the proportion of RNA corresponding to one or the other of the isoforms. To determine if there is a change in the expression of the D gene, one can proceed as indicated below.
La modification de l'expression du gène codant pour le récepteur dopaminergique D2 correspond soit à une surexpression du gène, notamment
sur les cellules lactotropes de l'hypophyse, par rapport à l'expression normale ci-dessus définie, soit à l'absence d'expression du gène D2.The modification of the expression of the gene coding for the dopaminergic receptor D 2 corresponds either to an overexpression of the gene, in particular on the pituitary cells of the pituitary gland, compared to the normal expression defined above, that is to say the absence of expression of the D 2 gene.
La surexpression de D2 peut être déterminée de la façon suivante.The overexpression of D 2 can be determined as follows.
On vérifie tout d'abord la présence d'un transgène. Un transgène est défini comme un fragment d'ADN qui est inséré de façon artificielle (c'est-à- dire autrement que par reproduction sexuelle) dans une cellule et qui devient partie intégrante au génome de l'animal qui se développe à partir de cette cellule, ou qui est modifié par rapport à l'ADN normalement présent dans la cellule. Un tel transgène peut être un gène homologue à un gène naturel de l'animal transgénique mais qui est inséré dans le génome de l'animal à une position qui diffère de celle de l'homologue naturel.We first check the presence of a transgene. A transgene is defined as a fragment of DNA which is inserted artificially (i.e. other than by sexual reproduction) in a cell and which becomes part of the genome of the animal which develops from that cell, or that is modified from the DNA normally present in the cell. Such a transgene may be a gene homologous to a natural gene of the transgenic animal but which is inserted into the genome of the animal at a position which differs from that of the natural homolog.
Pour vérifier la présence du transgène, on analyse l'ADN extrait d'un échantillon par exemple de queue de souris dérivé de l'injection du transgène dans des ovocytes fertilisés. Cet ADN est digéré par des enzymes de restriction, notamment PstI, et ensuite la présence du transgène est révélé par hybridation par exemple dans les conditions d'hybridation stringentes de cet ADN à une sonde marquée correspondant à une partie du transgène qui est hétérologue par rapport au génome de l'animal, par exemple au fragment de SV40 présent seulement dans la construction servie pour introduire le transgène, et par exemple le fragmentTo verify the presence of the transgene, the DNA extracted from a sample, for example from a mouse tail derived from the injection of the transgene into fertilized oocytes, is analyzed. This DNA is digested with restriction enzymes, in particular PstI, and then the presence of the transgene is revealed by hybridization, for example under the conditions of stringent hybridization of this DNA with a labeled probe corresponding to a part of the transgene which is heterologous with respect to to the animal's genome, for example to the fragment of SV40 present only in the construct used to introduce the transgene, and for example the fragment
BamHI-BglII de SV40.BamHI-BglII from SV40.
Les conditions d'hybridation sont les suivantes: les membranes de nylon sont hybridées dans une solution contenant: 50% de formamide, 5x SSC, 5x Denhardt's, 1 % SDS, ADN de sperme de saumon 100 mg/ml, pendant une nuit à 42°C. Les lavages sont effectués deux fois avec 2x SSC, 0,1 % SDS àThe hybridization conditions are as follows: the nylon membranes are hybridized in a solution containing: 50% formamide, 5x SSC, 5x Denhardt's, 1% SDS, salmon sperm DNA 100 mg / ml, overnight at 42 ° C. Washes are carried out twice with 2x SSC, 0.1% SDS at
42°C, puis deux fois avec 0,lx SSC à 65°C.42 ° C, then twice with 0.1 x SSC at 65 ° C.
Afin de vérifier que les souris contenant l'ADN transgénique expriment le transgène, on sacrifie l'animal transgénique ainsi qu'un animal contrôle et on prépare l'ARN à partir de différents tissus. On analyse l'ARN par la technique de transfert d'ARN (Northern blot) et on utilise comme sonde toujours la même sonde que celle définie ci-dessus, et notamment le même fragment de SV40 que celui qui a servi pour déterminer la présence du transgène.In order to verify that the mice containing the transgenic DNA express the transgene, the transgenic animal and a control animal are sacrificed and the RNA is prepared from different tissues. The RNA is analyzed by the RNA transfer technique (Northern blot) and the same probe always used as the probe defined above, and in particular the same fragment of SV40 as that which served to determine the presence of the transgene.
Une bande correspondant à l'expression du transgène est retrouvée seulement dans l'hypophyse des souris transgéniques et non pas dans l'animal contrôle.A band corresponding to the expression of the transgene is found only in the pituitary gland of the transgenic mice and not in the control animal.
Selon un mode de réalisation avantageux, l'invention concerne l'utilisation d'un animal mammifère transgénique non humain, susceptible de
surexprimer, notamment sur les cellules lactotropes de l'hypophyse, l' isoforme D2L et/ou l' isoforme D2S du récepteur dopaminergique D2.According to an advantageous embodiment, the invention relates to the use of a non-human transgenic mammal animal, capable of overexpressed, particularly on lactotrophs in the pituitary, isoform D 2 L and / or D isoform S 2 of the dopamine D 2 receptor.
Pour fixer les idées, la surexpression de D L par rapport à l'expression normale peut être d'environ 7 fois. Pour fixer les idées, la surexpression de D2S par rapport à l'expression normale peut être d'environ 10 fois.To fix the ideas, the overexpression of DL compared to the normal expression can be approximately 7 times. To fix the ideas, the overexpression of D 2 S compared to the normal expression can be approximately 10 times.
La surexpression est dépendante du promoteur utilisé, mais aussi du lieu d'insertion du transgène dans le génome. Ceci signifie que même en utilisant le même promoteur, on n'est pas sûr d'obtenir la même expression dans deux animaux indépendants, car dans un animal on peut avoir intégration du transgène dans une partie du génome plus active par rapport à l'autre, ou avoir plusieurs copies insérées dans un cas mais pas dans l'autre. C'est seulement un criblage entre plusieurs lignées transgéniques qui permet d'éventuellement sélectionner deux animaux avec des taux d'expression similaires pour le même gène ou pour deux gènes différents ayant le même promoteur.The overexpression is dependent on the promoter used, but also on the place of insertion of the transgene in the genome. This means that even using the same promoter, one is not sure of obtaining the same expression in two independent animals, because in one animal one can have integration of the transgene in a part of the genome more active compared to the other , or have multiple copies inserted in one case but not in the other. It is only a screening between several transgenic lines which makes it possible to possibly select two animals with similar levels of expression for the same gene or for two different genes having the same promoter.
La surexpression de l' isoforme D2L ou la surexpression de l' isoforme D S peut être déterminée en utilisant la technique de protection à la digestion par l'enzyme RNAse, en comparant l'intensité de la bande correspondant à chaque isoforme dans une souris contrôle et une souris transgénique. On soustrait la valeur de l'intensité de la bande obtenue avec des ARN provenant de souris contrôles (car il y a l'ARN normalement présent dans l'animal contrôle) à celle obtenue à partir de souris transgéniques.The overexpression of the D 2 L isoform or the overexpression of the DS isoform can be determined using the RNAse enzyme digestion protection technique, by comparing the intensity of the band corresponding to each isoform in a mouse. control and a transgenic mouse. The value of the intensity of the band obtained with RNA from control mice (since there is RNA normally present in the control animal) is subtracted from that obtained from transgenic mice.
Afin d'étudier la surexpression des protéines correspondantes aux récepteurs D L et D2S, on procède à des études de liaison (établissement des courbes de saturation: méthode de Scatchard (The attraction of proteins for small molécules and ions; _______ 1949, 51: 660) d'un agoniste ou d'un antagoniste marqué, de préférence la spipérone, à des homogénats de tissus provenant des hypophyses de souris transgéniques et normales. La comparaison entre les valeurs (nombre de sites) obtenues avec les deux groupes donne une indication de la surexpression du récepteur.In order to study the overexpression of the proteins corresponding to the DL and D 2 S receptors, binding studies are carried out (establishment of the saturation curves: Scatchard method (The attraction of proteins for small molecules and ions; _______ 1949, 51: 660) of a labeled agonist or antagonist, preferably spiperone, to tissue homogenates from the pituitaries of transgenic and normal mice. A comparison between the values (number of sites) obtained with the two groups gives an indication receptor overexpression.
Selon un mode de réalisation, l'invention concerne l'utilisation d'un animal mammifère transgénique non humain qui n'exprime plus l' isoforme D L, ou qui n'exprime plus ni l'isoforme D L, ni l'isoforme D2S.According to one embodiment, the invention relates to the use of a non-human transgenic mammalian animal which no longer expresses the DL isoform, or which no longer expresses either the DL isoform or the D 2 S isoform. .
L'absence d'expression de l'isoforme D2L peut être déterminée de la façon suivante.The absence of expression of the D 2 L isoform can be determined as follows.
Des souris où l'expression de la forme D L a été éliminée sont d'abord identifiées relativement à l'ADN par analyse avec la méthode de transfert d'ADN (Southern blot), à l'aide d'une sonde consistant en un fragment
contenant tout ou partie de la région du récepteur D2 qui contient l'insertion de 87 paires de base définie ci-dessus, notamment une sonde génomique montrée dans la figure 4, EcoRI-BglI.Mice where expression of the DL form has been eliminated are first identified relative to the DNA by analysis with the DNA transfer method (Southern blot), using a probe consisting of a fragment. containing all or part of the D 2 receptor region which contains the insertion of 87 base pairs defined above, in particular a genomic probe shown in FIG. 4, EcoRI-BglI.
Afin d'analyser que l'expression de la forme D2S est toujours présente dans ces animaux, on analyse l'ARN extrait à partir du striatum et de l'hypophyse ainsi que d'autres tissus, par exemple la substantia nigra, par la technique de protection à la digestion par l'enzyme RNAse.In order to analyze that the expression of the D 2 S form is still present in these animals, the RNA extracted from the striatum and the pituitary gland as well as other tissues, for example the substantia nigra, is analyzed by the technique of protection against digestion by the enzyme RNAse.
On utilise aussi des tissus qui n'expriment pas le récepteur et qui servent de contrôle comme par exemple, le cervelet. Afin de vérifier que la protéine D2S est toujours présente, on analyse les propriétés pharmacologiques par liaison d'un ligand D2 spécifique, par exemple dans les homogénats de striatum ou d'hypophyse.We also use tissues that do not express the receptor and that serve as controls, such as the cerebellum. In order to verify that the protein D 2 S is still present, the pharmacological properties are analyzed by binding of a specific ligand D 2 , for example in striatum or pituitary homogenates.
L'absence d'expression de l'isoforme D2L et de D2S peut être déterminée par l'une des méthodes suivantes: 1) Des souris où l'expression de la forme D2L et D2S a été éliminée, sont vérifiées par transfert d'ADN (Southern blot) avec de l'ADN extrait de la queue de souris coupé avec l'enzyme Spel et hybride avec la sonde PstI correspondant au fragment indiqué dans la figure 3.The absence of expression of the D 2 L isoform and of D 2 S can be determined by one of the following methods: 1) Mice in which the expression of the form D 2 L and D 2 S has been eliminated , are verified by DNA transfer (Southern blot) with DNA extracted from the mouse tail cut with the Spel enzyme and hybridized with the PstI probe corresponding to the fragment indicated in FIG. 3.
L'hybridation de cette sonde montre clairement que la bande correspondant au récepteur D de type sauvage (c'est-à-dire normalement présent chez les animaux) n'est plus présente dans les animaux homozygotes pour la mutation.Hybridization of this probe clearly shows that the band corresponding to the wild type D receptor (that is to say normally present in animals) is no longer present in animals homozygous for the mutation.
2) L'analyse par transfert d'ARN (Northern blot) des ARN extraits des tissus exprimant D2, notamment striatum, montre qu'il n'y a plus d'expression d'ARN correspondant au gène sauvage. La sonde utilisée dans ce cas comprend la partie 5' de l'ADNc du gène D2, en particulier on peut utiliser un fragment Smal-PvuII de 454 paires de bases (Figure 9A).2) Analysis by RNA transfer (Northern blot) of RNA extracted from tissues expressing D 2 , in particular striatum, shows that there is no longer any expression of RNA corresponding to the wild-type gene. The probe used in this case comprises the 5 ′ part of the cDNA of the D 2 gene, in particular a SmaI-PvuII fragment of 454 base pairs can be used (FIG. 9A).
3) Enfin on peut démontrer aussi que la liaison de ligands spécifiques D , notamment spipérone, est complètement absente dans ces souris (Figure 9B, cercles blancs).3) Finally, it can also be demonstrated that the binding of specific ligands D, in particular spiperone, is completely absent in these mice (FIG. 9B, white circles).
Selon un mode de réalisation avantageux, l'invention concerne un animal mammifère transgénique non humain ou cellules de mammifères contenant dans leur génome une construction choisie parmi:According to an advantageous embodiment, the invention relates to a non-human transgenic mammal animal or mammalian cells containing in their genome a construct chosen from:
- un promoteur permettant la surexpression de l'isoforme D2L sur les cellules lactotropes de l'hypophyse, notamment le promoteur de la prolactine,a promoter allowing the overexpression of the D 2 L isoform on the lactotropic cells of the pituitary gland, in particular the prolactin promoter,
+ ADNc de D2L + un terminateur de transcription, notamment une partie du génome du virus SV40 contenant le site de polyadénylation;
- un promoteur permettant la surexpression de l'isoforme D S sur les cellules lactotropes de l'hypophyse, notamment le promoteur de la prolactine, + ADNc de D2S + un terminateur de transcription, notamment une partie du génome du virus SV40 contenant le site de polyadénylation; - un promoteur permettant la surexpression de l'isoforme D2L sur les cellules lactotropes de l'hypophyse, notamment le promoteur de la prolactine, + ADNc de D2L + un terminateur de transcription, notamment une partie du génome du virus SV40 contenant le site de polyadénylation, et un promoteur permettant la surexpression de l'isoforme D S sur les cellules lactotropes de l'hypophyse, notamment le promoteur de la prolactine, + ADNc de D2S + un terminateur de transcription, notamment une partie du génome du virus SV40 contenant le site de polyadénylation.+ D 2 L cDNA + a transcription terminator, in particular a part of the SV40 virus genome containing the polyadenylation site; a promoter allowing the overexpression of the DS isoform on the lactotropic cells of the pituitary gland, in particular the prolactin promoter, + cDNA of D 2 S + a transcription terminator, in particular a part of the genome of the SV40 virus containing the site polyadenylation; - a promoter allowing the overexpression of the D 2 L isoform on the lactotropic cells of the pituitary gland, in particular the prolactin promoter, + D 2 L cDNA + a transcription terminator, in particular a part of the genome of the SV40 virus containing the polyadenylation site, and a promoter allowing the overexpression of the DS isoform on the lactotropic cells of the pituitary gland, in particular the prolactin promoter, + D 2 S cDNA + a transcription terminator, in particular a part of the genome of the SV40 virus containing the polyadenylation site.
Par ADNc de D L ou ADNc de D S, on englobe également toute séquence nucléotidique dérivée des séquences respectivement représentées sur les figures 5 et 6, à savoir par addition, délétion ou mutation d'une ou plusieurs bases sous réserve que la protéine codée correspondante à cette séquence nucléotidique présente des propriétés biologiques équivalentes à celles des récepteurs D .The term “cDNA of DL or cDNA of DS” also includes any nucleotide sequence derived from the sequences represented respectively in FIGS. 5 and 6, namely by addition, deletion or mutation of one or more bases, provided that the coded protein corresponding to this nucleotide sequence has biological properties equivalent to those of D receptors.
Par ADNc de D L ou de D S, on désigne également toute séquence nucléotidique susceptible d'hybrider respectivement avec les séquences représentées sur les figures 5 et 6, ou en tenant compte de la dégénérescence du code génétique, susceptible de coder pour les protéines respectivement représentées sur les figures 5 et 6.By DL or DS cDNA, we also designate any nucleotide sequence capable of hybridizing respectively with the sequences represented in FIGS. 5 and 6, or taking into account the degeneration of the genetic code, capable of coding for the proteins respectively represented on Figures 5 and 6.
Les animaux mammifères transgéniques non humains sont tels qu'il y a surexpression de D L et/ou D2S sur les cellules lactotropes de l'hypophyse.Non-human transgenic mammalian animals are such that there is overexpression of DL and / or D 2 S on the lactotropic cells of the pituitary gland.
L'invention concerne un animal mammifère transgénique non humain ou des cellules de mammifères contenant le gène du récepteur D dans lequel un fragment du gène de D2 contenant l'exon 2 ou un fragment du gène de D contenant l'exon 6 est remplacé par tout ou partie d'un gène marqueur sous le contrôle d'un promoteur approprié, notamment le gène de résistance à la néomycine (neo) sous le contrôle du promoteur phosphoglycérate kinase -1 (PGK-1).The invention relates to a non-human transgenic mammal animal or mammalian cells containing the D receptor gene in which a fragment of the D 2 gene containing exon 2 or a fragment of the D gene containing exon 6 is replaced by all or part of a marker gene under the control of an appropriate promoter, in particular the neomycin resistance gene (neo) under the control of the phosphoglycerate kinase -1 promoter (PGK-1).
Dans le cas d'un animal mammifère transgénique non humain dont le gène du récepteur D2 est tel qu'un fragment contenant l'exon 2 est supprimé, l'expression de D L et de D2S est supprimée.In the case of a non-human transgenic mammalian animal whose D 2 receptor gene is such that a fragment containing exon 2 is deleted, the expression of DL and of D 2 S is suppressed.
Dans le cas d'un animal mammifère transgénique non humain dont le gène du récepteur D2 est tel qu'un fragment contenant l'exon 6 est supprimé, l'expression de D L est supprimée, mais l'expression de D S est maintenue.
L'invention concerne également les constructions transgéniques choisies parmi: a) un promoteur permettant la surexpression de l'isoforme D2L sur les cellules lactotropes de l'hypophyse, notamment le promoteur de la prolactine, + ADNc de D2L + un terminateur de transcription, notamment une partie du génome du virus SV40 contenant le site de polyadénylation et en particulier: fragment BamHI-HindII(-3000/-l-33) du promoteur de la prolactine + fragment Smal-EcoRV de l'ADNc de D2L + fragment BamHI-BglII de SV40, b) un promoteur permettant la surexpression de l'isoforme D2S sur les cellules lactotropes de l'hypophyse, notamment le promoteur de la prolactine,In the case of a non-human transgenic mammalian animal whose D 2 receptor gene is such that a fragment containing exon 6 is deleted, the expression of DL is suppressed, but the expression of DS is maintained. The invention also relates to the transgenic constructs chosen from: a) a promoter allowing the overexpression of the D 2 L isoform on the lactotropic cells of the pituitary gland, in particular the prolactin promoter, + D 2 L cDNA + a terminator of transcription, in particular a part of the SV40 virus genome containing the polyadenylation site and in particular: BamHI-HindII fragment (-3000 / -l-33) of the prolactin promoter + Smal-EcoRV fragment of the D 2 cDNA L + BamHI-BglII fragment of SV40, b) a promoter allowing the overexpression of the isoform D 2 S on the lactotropic cells of the pituitary gland, in particular the prolactin promoter,
+ ADNc de D2S + un terminateur de transcription, notamment une partie du génome du virus SV40 contenant le site de polyadénylation et en particulier: fragment BamHI-HindII(-3000/+33) du promoteur de la prolactine + fragment Smal-EcoRV de l'ADNc de D2S + fragment BamHI-BglII de SV40, c) un promoteur permettant la surexpression de l'isoforme D L sur les cellules lactotropes de l'hypophyse, notamment le promoteur de la prolactine, + ADNc de D2L + un terminateur de transcription, notamment une partie du génome du virus SV40 contenant le site de polyadénylation et en particulier: fragment BamHI-HindII(-3000/+33) du promoteur de la prolactine + fragment Smal-EcoRV de l'ADNc de D2L + fragment BamHI-BglII de SV40, et un promoteur permettant la surexpression de l'isoforme D2S sur les cellules lactotropes de l'hypophyse, notamment le promoteur de la prolactine, + ADNc de D S + un terminateur de transcription, notamment une partie du génome du virus SV40 contenant le site de polyadénylation et en particulier: fragment BamHI-HindII(-3000/+33) du promoteur de la prolactine + fragment Smal-EcoRV de l'ADNc de D2S + fragment BamHI-BglII de SV40, d) le gène de D2 dans lequel un fragment contenant l'exon 2 est remplacé par un gène marqueur sous le contrôle d'un promoteur approprié, notamment une cassette contenant le gène de la résistance à la néomycine (neo) sous le contrôle du promoteur PGKI, et en particulier le gène de D2 dans lequel le fragment génomique de 0,9 Kb Ncol, contenant l'exon 2 est remplacé par la cassette PGK-neo. e) le gène de D2 dans lequel un fragment contenant l'exon 6 est remplacé par un gène marqueur sous le contrôle d'un promoteur approprié, notamment par une cassette contenant le gène neo sous le contrôle du promoteur PGKI, et en particulier le gène de D2 dans lequel le fragment génomique BglII-BglII contenant l'exon 6 est remplacé par la cassette PGK- neo.
Selon un mode de réalisation avantageux, l'invention concerne des cultures cellulaires contenant l'une des susdites constructions transgéniques.+ D 2 S cDNA + a transcription terminator, in particular a part of the SV40 virus genome containing the polyadenylation site and in particular: BamHI-HindII fragment (-3000 / + 33) of the prolactin promoter + Smal-EcoRV fragment of the D 2 S cDNA + BamHI-BglII fragment of SV40, c) a promoter allowing the overexpression of the DL isoform on the lactotropic cells of the pituitary gland, in particular the prolactin promoter, + D 2 L cDNA + a transcription terminator, in particular a part of the genome of the SV40 virus containing the polyadenylation site and in particular: BamHI-HindII fragment (-3000 / + 33) of the prolactin promoter + Smal-EcoRV fragment of D cDNA 2 L + BamHI-BglII fragment of SV40, and a promoter allowing the overexpression of the D 2 S isoform on the lactotropic cells of the pituitary gland, in particular the prolactin promoter, + DS cDNA + a transcription terminator, in particular part of the SV40 virus genome containing the polyadé site nylation and in particular: BamHI-HindII fragment (-3000 / + 33) of the prolactin promoter + Smal-EcoRV fragment of the D 2 S cDNA + BamHI-BglII fragment of SV40, d) the D 2 gene in which a fragment containing exon 2 is replaced by a marker gene under the control of an appropriate promoter, in particular a cassette containing the gene for resistance to neomycin (neo) under the control of the promoter PGKI, and in particular the gene of D 2 in which the 0.9 Kb Ncol genomic fragment containing exon 2 is replaced by the PGK-neo cassette. e) the D 2 gene in which a fragment containing exon 6 is replaced by a marker gene under the control of an appropriate promoter, in particular by a cassette containing the neo gene under the control of the PGKI promoter, and in particular the D 2 gene in which the BglII-BglII genomic fragment containing exon 6 is replaced by the PGK-neo cassette. According to an advantageous embodiment, the invention relates to cell cultures containing one of the above transgenic constructs.
Ces cultures cellulaires peuvent être obtenues soit à partir de cellules prélevées sur des animaux transgéniques tels que définis ci-dessus ou soit à partir de lignées cellulaires en utilisant les susdites constructions transgéniques à l'aide de techniques standard de transfection cellulaire.These cell cultures can be obtained either from cells taken from transgenic animals as defined above or either from cell lines using the above transgenic constructs using standard cell transfection techniques.
L'invention concerne également des cultures cellulaires, dans lesquelles les cellules sont telles que l'on a enlevé sur l'un des deux allèles l'exon 2, ou l'exon 6 du gène de D . L'invention concerne également un mammifère transgénique non humain tel qu'obtenu par introduction, notamment microinjection dans un ovocyte fécondé, de l'une des susdites constructions transgéniques, insertion de l'embryon dans une mère de substitution et développement de l'embryon à terme. Selon un mode de réalisation avantageux de l'invention, les susdits mammifères transgéniques non humains contiennent l'une des constructions a), b) ou c), telles que définies précédemment.The invention also relates to cell cultures, in which the cells are such that exon 2 or exon 6 of the D gene has been removed from one of the two alleles. The invention also relates to a non-human transgenic mammal as obtained by introduction, in particular microinjection into a fertilized oocyte, of one of the above transgenic constructions, insertion of the embryo into a surrogate mother and development of the embryo to term. According to an advantageous embodiment of the invention, the above non-human transgenic mammals contain one of the constructions a), b) or c), as defined above.
L'invention concerne également un mammifère transgénique non humain tel qu'obtenu par introduction dans un blastocyte de cellules souches embryonnaires (cellules ES) comportant dans leur génome l'une des susdites constructions transgéniques, obtenue par recombinaison homologue, sélection d'animaux chimères mâles selon un critère correspondant à la lignée ES, croisement des animaux sélectionnés avec des souris, notamment des souris C57 Black 6, pour obtenir des animaux hétérozygotes par rapport à l'une des susdites constructions et éventuellement croisement de deux hétérozygotes pour obtenir un animal homozygote par rapport à l'une des susdites constructions.The invention also relates to a non-human transgenic mammal as obtained by introduction into a blastocyte of embryonic stem cells (ES cells) comprising in their genome one of the above-mentioned transgenic constructions, obtained by homologous recombination, selection of male chimeric animals according to a criterion corresponding to the ES line, crossing of the selected animals with mice, in particular C57 Black 6 mice, to obtain animals heterozygous with respect to one of the above-mentioned constructions and possibly crossing of two heterozygotes to obtain an animal homozygous with in relation to one of the above constructions.
On choisit de façon avantageuse les souris C57 Black 6, car elles sont classiquement utilisées dans des expériences de comportement. De façon avantageuse, les constructions visées dans les susdits animaux transgéniques sont les constructions d) et e), telles que définies précédemment. L'invention concerne également un mammifère transgénique tel que produit par croisement d'animaux transgéniques exprimant l'une des constructions transgéniques définies ci-dessus. En particulier, les animaux transgéniques contenant la construction c), peuvent être obtenus soit par introduction des constructions a) et b) dans leur génome, soit par croisement d'un animal transgénique contenant la
construction a) avec un animal transgénique contenant la construction transgénique b).C57 Black 6 mice are advantageously chosen, because they are conventionally used in behavioral experiments. Advantageously, the constructions referred to in the above transgenic animals are the constructions d) and e), as defined above. The invention also relates to a transgenic mammal as produced by crossing transgenic animals expressing one of the transgenic constructs defined above. In particular, the transgenic animals containing construct c) can be obtained either by introducing the constructs a) and b) into their genome, or by crossing a transgenic animal containing the construction a) with a transgenic animal containing the transgenic construction b).
L'invention concerne également un procédé d'obtention d'un modèle transgénique pour l'étude des pathologies impliquant les récepteurs D2, et de leur traitement, comprenant l'introduction dans les cellules ou des embryons de mammifères non humains d'une construction choisie parmi:The invention also relates to a method for obtaining a transgenic model for the study of pathologies involving D 2 receptors, and to their treatment, comprising the introduction into the cells or embryos of non-human mammals of a construct chosen from:
- un promoteur permettant la surexpression de l'isoforme D2L sur les cellules lactotropes de l'hypophyse, notamment le promoteur de la prolactine, + ADNc de D2L + un terminateur de transcription, notamment une partie du génome du virus SV40 contenant le site de polyadénylation et en particulier: fragment BamHI-HindII(-30O0/+33) du promoteur de la prolactine + fragment Smal-EcoRV de l'ADNc de D2L + fragment BamHI-BglII de SV40,- a promoter allowing the overexpression of the D 2 L isoform on the lactotropic cells of the pituitary gland, in particular the prolactin promoter, + D 2 L cDNA + a transcription terminator, in particular a part of the genome of the SV40 virus containing the polyadenylation site and in particular: BamHI-HindII fragment (-30O0 / + 33) of the prolactin promoter + Smal-EcoRV fragment of D 2 L cDNA + BamHI-BglII fragment of SV40,
- un promoteur permettant la surexpression de l'isoforme D2S sur les cellules lactotropes de l'hypophyse, notamment le promoteur de la prolactine, + ADNc de D2S + un terminateur de transcription, notamment une partie du génome du virus SV40 contenant le site de polyadénylation et en particulier: fragment BamHI-HindII(-3000/+33) du promoteur de la prolactine + fragment Smal-EcoRV de l'ADNc de D2S -I- fragment BamHI-BglII de SV40,- a promoter allowing the overexpression of the isoform D 2 S on the lactotropic cells of the pituitary gland, in particular the promoter of prolactin, + cDNA of D 2 S + a transcription terminator, in particular a part of the genome of the SV40 virus containing the polyadenylation site and in particular: BamHI-HindII fragment (-3000 / + 33) of the prolactin promoter + SmaI-EcoRV fragment of the D 2 S cDNA -I- BamHI-BglII fragment of SV40,
- un promoteur permettant la surexpression de l'isoforme D2L sur les cellules lactotropes de l'hypophyse, notamment le promoteur de la prolactine,a promoter allowing the overexpression of the D 2 L isoform on the lactotropic cells of the pituitary gland, in particular the prolactin promoter,
+ ADNc de D2L + un terminateur de transcription, notamment une partie du génome du virus SV40 contenant le site de polyadénylation et en particulier: fragment BamHI-HindII(-3000/+33) du promoteur de la prolactine, + fragment Smal-EcoRV de l'ADNc de D2L + fragment BamHI-BglII de SV40, - et un promoteur permettant la surexpression de l'isoforme D2S sur les cellules lactotropes de l'hypophyse, notamment le promoteur de la prolactine, + ADNc de D2S + un terminatc . de transcription, notamment une partie du génome du virus SV40 contenant le site de polyadénylation et en particulier: fragment BamHI-HindII(-3000/+33) du promoteur de la prolactine 4- fragment Smal-EcoRV de l'ADNc de D2S + fragment BamHI-BglII de SV40,+ D 2 L cDNA + a transcription terminator, in particular a part of the SV40 virus genome containing the polyadenylation site and in particular: BamHI-HindII fragment (-3000 / + 33) of the prolactin promoter, + Smal fragment EcoRV of the D 2 L cDNA + BamHI-BglII fragment of SV40, - and a promoter allowing the overexpression of the D 2 S isoform on the lactotropic cells of the pituitary gland, in particular the prolactin promoter, + cDNA of D 2 S + a terminatc. of transcription, in particular a part of the genome of the SV40 virus containing the polyadenylation site and in particular: BamHI-HindII fragment (-3000 / + 33) of the prolactin promoter 4- Smal-EcoRV fragment of the D 2 S cDNA + BamHI-BglII fragment of SV40,
- le gène de D2 dans lequel un fragment contenant l'exon 2 est remplacé par un gène marqueur sous le contrôle d'un promoteur approprié, notamment une cassette contenant le gène neo sous le contrôle du promoteur PGKI, et en particulier le gène de D2 dans lequel le fragment génomique de 0,9 Kb Ncol, contenant l'exon 2 est remplacé par la cassette PGK-neo.the gene for D 2 in which a fragment containing exon 2 is replaced by a marker gene under the control of an appropriate promoter, in particular a cassette containing the neo gene under the control of the PGKI promoter, and in particular the gene for D 2 in which the 0.9 Kb Ncol genomic fragment containing exon 2 is replaced by the PGK-neo cassette.
- le gène de D2 dans lequel un fragment contenant l'exon 6 est remplacé par un gène marqueur sous le contrôle d'un promoteur approprié, notamment par une cassette contenant le gène neo sous le contrôle du promoteur PGKI, et
en particulier le gène de D2 dans lequel le fragment génomique BglII-BglII contenant l'exon 6 est remplacé par la cassette PGK-neo.the D 2 gene in which a fragment containing exon 6 is replaced by a marker gene under the control of an appropriate promoter, in particular by a cassette containing the neo gene under the control of the PGKI promoter, and in particular the D 2 gene in which the BglII-BglII genomic fragment containing exon 6 is replaced by the PGK-neo cassette.
L'invention concerne également un procédé de criblage de médicaments actifs sur des pathologies impliquant les récepteurs D2, notamment les prolactinomes, ou certaines pathologies neurodégénératives telles que la maladie de Parkinson, la maladie d'Huntington ou la schizophrénie, comprenant:The invention also relates to a method for screening for drugs active on pathologies involving D 2 receptors, in particular prolactinomas, or certain neurodegenerative pathologies such as Parkinson's disease, Huntington's disease or schizophrenia, comprising:
* l'administration à un mammifère non humain transgénique ou à des cellules de mammifères non humains transgéniques contenant une construction choisie parmi:* administration to a transgenic non-human mammal or to cells of transgenic non-human mammals containing a construct chosen from:
- un promoteur permettant la surexpression de l'isoforme D2L sur les cellules lactotropes de l'hypophyse, notamment le promoteur de la prolactine, + ADNc de D2L + un terminateur de transcription, notamment une partie du génome du virus SV40 contenant le site de polyadénylation et en particulier: fragment BamHI-HindII(-3000/+33) du promoteur de la prolactine + fragment Smal-EcoRV de l'ADNc de D2L + fragment BamHI-BglII de SV40,- a promoter allowing the overexpression of the D 2 L isoform on the lactotropic cells of the pituitary gland, in particular the prolactin promoter, + D 2 L cDNA + a transcription terminator, in particular a part of the genome of the SV40 virus containing the polyadenylation site and in particular: BamHI-HindII fragment (-3000 / + 33) of the prolactin promoter + Smal-EcoRV fragment of the D 2 L cDNA + BamHI-BglII fragment of SV40,
- un promoteur permettant la surexpression de l'isoforme D2S sur les cellules lactotropes de l'hypophyse, notamment le promoteur de la prolactine, -I- ADNc de D2S + un terminateur de transcription, notamment une partie du génome du virus SV40 contenant le site de polyadénylation et en particulier: fragment BamHI-HïndII(-30O0/+33) du promoteur de la prolactine + fragment Smal-EcoRV de l'ADNc de D2S + fragment BamHI-BglII de SV40,a promoter allowing the overexpression of the isoform D 2 S on the lactotropic cells of the pituitary gland, in particular the promoter of prolactin, -I- cDNA of D 2 S + a transcription terminator, in particular a part of the genome of the virus SV40 containing the polyadenylation site and in particular: BamHI-HindII fragment (-30O0 / + 33) of the prolactin promoter + Smal-EcoRV fragment of D 2 S cDNA + BamHI-BglII fragment of SV40,
- un promoteur permettant la surexpression de l'isoforme D L sur les cellules lactotropes de l'hypophyse, notamment le promoteur de la prolactine, + ADNc de D2L + un terminateur de transcription, notamment une partie du génome du virus SV40 contenant le site de polyadénylation et en particulier: fragment BamHI-HindII(-3000/+33) du promoteur de la prolactine + fragment Smal-EcoRV de l'ADNc de D L + fragment BamHI-BglII de SV40,a promoter allowing the overexpression of the DL isoform on the lactotropic cells of the pituitary gland, in particular the prolactin promoter, + cDNA of D 2 L + a transcription terminator, in particular a part of the genome of the SV40 virus containing the site polyadenylation and in particular: BamHI-HindII fragment (-3000 / + 33) of the prolactin promoter + SmaI-EcoRV fragment of DL cDNA + BamHI-BglII fragment of SV40,
- et un promoteur permettant la surexpression de l'isoforme D S sur les cellules lactotropes de l'hypophyse, notamment le promoteur de la prolactine,- and a promoter allowing the overexpression of the isoform D S on the lactotropic cells of the pituitary gland, in particular the prolactin promoter,
+ ADNc de D S + un terminateur de transcription, notamment une partie du génome du virus SV40 contenant le site de polyadénylation et en particulier: fragment BamHI-HindII(-3000/+33) du promoteur de la prolactine + fragment Smal-EcoRV de l'ADNc de D2S + fragment BamHI-BglII de SV40, - le gène de D2 dans lequel un fragment contenant l'exon 2 est remplacé par un gène marqueur sous le contrôle d'un promoteur approprié, notamment une cassette contenant le gène neo sous le contrôle du promoteur PGKI, et en
particulier le gène de D2 dans lequel le fragment génomique de 0,9 Kb Ncol, contenant l'exon 2 est remplacé par la cassette PGK-neo,+ DS cDNA + a transcription terminator, in particular a part of the SV40 virus genome containing the polyadenylation site and in particular: BamHI-HindII fragment (-3000 / + 33) of the prolactin promoter + Smal-EcoRV fragment of the CDNA of D 2 S + BamHI-BglII fragment of SV40, - the D 2 gene in which a fragment containing exon 2 is replaced by a marker gene under the control of an appropriate promoter, in particular a cassette containing the gene neo under the control of the promoter PGKI, and in particular the D 2 gene in which the genomic fragment of 0.9 Kb Ncol, containing exon 2 is replaced by the PGK-neo cassette,
- le gène de D2 dans lequel un fragment contenant l'exon 6 est remplacé par un gène marqueur sous le contrôle d'un promoteur approprié, notamment par une cassette contenant le gène neo sous le contrôle du promoteur PGKI, et en particulier le gène de D2 dans lequel le fragment génomique Bgiπ-BglH contenant l'exon 6 est remplacé par la cassette PGK-neo du composé à tester,- the D 2 gene in which a fragment containing exon 6 is replaced by a marker gene under the control of an appropriate promoter, in particular by a cassette containing the neo gene under the control of the PGKI promoter, and in particular the gene D 2 in which the Bgiπ-BglH genomic fragment containing exon 6 is replaced by the PGK-neo cassette of the compound to be tested,
* et la détermination de la diminution du taux de prolactine (dans le cas des prolactinomes) ou de l'amélioration de l'état neurodégénératif (dans le cas des pathologies neurodégénératives).* and the determination of the decrease in the level of prolactin (in the case of prolactinomas) or the improvement of the neurodegenerative state (in the case of neurodegenerative pathologies).
Les prolactinomes sont des tumeurs très fréquentes chez la femme. On ne connaît pas encore les origines de ces tumeurs, mais un mauvais fonctionnement du contrôle dopaminergique au niveau des lactotropes est impliqué. En particulier les animaux transgéniques de l'invention pourront servir de modèle pour l'étude et la détermination du traitement d'une telle pathologie. En effet, dans ces animaux, l'équilibre normal entre l'expression de la forme D2L et D2S est altéré, ce qui permet d'étudier le phénotype correspondant à une telle altération. En même temps, des agonistes et antagonistes dopaminergiques D2 peuvent être testés dans leur efficacité à augmenter ou supprimer les phénotypes obtenus.Prolactinomas are very common tumors in women. The origins of these tumors are not yet known, but a malfunction of dopaminergic control at the lactotropic level is implicated. In particular, the transgenic animals of the invention may serve as a model for the study and the determination of the treatment of such a pathology. Indeed, in these animals, the normal balance between the expression of the form D 2 L and D 2 S is altered, which makes it possible to study the phenotype corresponding to such an alteration. At the same time, dopamine D 2 agonists and antagonists can be tested for their effectiveness in increasing or suppressing the phenotypes obtained.
Par exemple la surexpression de la forme D2S provoque une diminution importante du taux de prolactine avec pour conséquence un déficit de lactation chez les femelles. Le remoxipride est un antagoniste connu pour avoir une forte spécificité de liaison avec D2S, ce qui permet de tester si l'injection de cette substance permet d'abolir le déficit du taux de prolactine chez les animaux transgéniques surexprimant D2S.For example, the overexpression of the D 2 S form causes a significant decrease in the level of prolactin with the consequence of a lactation deficit in females. Remoxipride is an antagonist known to have a high specificity for binding with D 2 S, which makes it possible to test whether the injection of this substance makes it possible to eliminate the deficit in the level of prolactin in transgenic animals overexpressing D 2 S.
S 'agissant des animaux qui n'expriment plus ni D2S, ni D2L, ils peuvent servir de modèle pour l'étude du traitement de certaines maladies neurodégénératives .As regards animals which no longer express either D 2 S or D 2 L, they can serve as a model for studying the treatment of certain neurodegenerative diseases.
Les récepteurs D2 sont impliqués dans le contrôle de plusieurs fonctions physiologiques telles que la coordination du mouvement, la vision, la régulation de la synthèse de certaines hormones etc. On pourra donc mettre en évidence les déficits liés à l'absence de ces récepteurs. Par exemple, un des phénotypes liés à l'absence du récepteur D2 peut être une mauvaise coordination des mouvements. Ces animaux peuvent alors servir pour tester
des composés agissant au niveau de la stimulation du mouvement, afin de voir si ce déficit peut être compensé.D 2 receptors are involved in the control of several physiological functions such as movement coordination, vision, regulation of the synthesis of certain hormones, etc. We can therefore highlight the deficits linked to the absence of these receptors. For example, one of the phenotypes linked to the absence of the D 2 receptor can be poor coordination of movements. These animals can then be used to test compounds acting on the stimulation of movement, in order to see if this deficit can be compensated.
Du point de vue pharmacologique, les animaux transgéniques de l'invention sont intéressants pour cribler les substances actuellement utilisées avec une spécificité D afin de définir si tel est vraiment le cas. Comme il existe in vivo, plusieurs récepteurs dopaminergiques qui présentent des affinités très voisines pour les agonistes et antagonistes dopaminergiques existant; les animaux transgéniques de l'invention constituent donc un outil remarquable dans ce sens. S 'agissant des animaux transgéniques qui n'expriment plus que la formeFrom the pharmacological point of view, the transgenic animals of the invention are advantageous for screening the substances currently used with specificity D in order to define if this is really the case. As there are in vivo, several dopaminergic receptors which have very similar affinities for the existing dopamine agonists and antagonists; the transgenic animals of the invention therefore constitute a remarkable tool in this sense. As for transgenic animals which only express the form
D2S du récepteur, la recherche de phénotypes peut être faite et on pourra après songer aux possibles traitements.D 2 S of the receptor, the search for phenotypes can be made and we can then think about possible treatments.
De plus, les animaux transgéniques de l'invention n'exprimant plus que la forme D S du récepteur, peuvent être utilisés pour développer des antagonistes D2S spécifiques, avec la possibilité de pouvoir tester ces antagonistes in vivo en absence du récepteur D2L.In addition, the transgenic animals of the invention expressing only the DS form of the receptor can be used to develop specific D 2 S antagonists, with the possibility of being able to test these antagonists in vivo in the absence of the D 2 L receptor. .
L'invention concerne un procédé de criblage de médicaments actifs sur un modèle animal où les taux de prolactine sont diminués par rapport à l'état normal à l'aide d'animaux transgéniques surexprimant D2S, comprenant: * l'administration à un mammifère non humain transgénique ou à des cellules de mammifères non humains transgéniques contenant une construction choisie parmi:The invention relates to a method for screening for drugs active in an animal model in which the prolactin levels are reduced compared to the normal state using transgenic animals overexpressing D 2 S, comprising: * administration to a transgenic non-human mammal or to transgenic non-human mammal cells containing a construct chosen from:
- un promoteur permettant la surexpression de l'isoforme D2S sur les cellules lactotropes de l'hypophyse, notamment le promoteur de la prolactine, + ADNc de D2S + un terminateur de transcription, notamment une partie du génome du virus SV40 contenant le site de polyadénylation et en particulier: fragment BamHI-HindII(-3000/+33) du promoteur de la prolactine + fragment Smal-EcoRV de l'ADNc de D2S + fragment BamHI-BglII de SV40, du composé à tester, * et la détermination de l'augmentation du taux de prolactine.- a promoter allowing the overexpression of the isoform D 2 S on the lactotropic cells of the pituitary gland, in particular the promoter of prolactin, + cDNA of D 2 S + a transcription terminator, in particular a part of the genome of the SV40 virus containing the polyadenylation site and in particular: BamHI-HindII fragment (-3000 / + 33) of the prolactin promoter + Smal-EcoRV fragment of the D 2 S cDNA + BamHI-BglII fragment of SV40, of the compound to be tested, * and the determination of the increase in prolactin level.
L'invention concerne un procédé de criblage de médicaments actifs sur des prolactinomes à l'aide d'animaux transgéniques surexprimant D L, comprenant:The invention relates to a method for screening for drugs active on prolactinomas using transgenic animals overexpressing D L, comprising:
* l'administration à un mammifère non humain transgénique ou à des cellules de mammifères non humains transgéniques contenant une construction choisie parmi:* administration to a transgenic non-human mammal or to cells of transgenic non-human mammals containing a construct chosen from:
- un promoteur permettant la surexpression de l'isoforme D2L sur les cellules lactotropes de l'hypophyse, notamment le promoteur de la prolactine,
+ ADNc de D2L + un terminateur de transcription, notamment une partie du génome du virus SV40 contenant le site de polyadénylation et en particulier: fragment BamHI-HindII(-3000/+33) du promoteur de la prolactine + fragment Smal-EcoRV de l'ADNc de D2L + fragment BamHI-BglII de SV40, du composé à tester,a promoter allowing the overexpression of the D 2 L isoform on the lactotropic cells of the pituitary gland, in particular the prolactin promoter, + D 2 L cDNA + a transcription terminator, in particular a part of the SV40 virus genome containing the polyadenylation site and in particular: BamHI-HindII fragment (-3000 / + 33) of the prolactin promoter + Smal-EcoRV fragment cDNA of D 2 L + fragment BamHI-BglII of SV40, of the compound to be tested,
* et la détermination de la diminution du taux de prolactine. L'invention concerne un procédé de criblage de médicaments actifs sur des maladies neurodégénératives telles que la maladie de Parkinson, la maladie d'Huntington ou la schizophrénie à l'aide d'animaux transgéniques dans lesquels D2S et D L ne sont plus exprimés, comprenant* and the determination of the decrease in prolactin level. The invention relates to a method for screening for drugs which are active on neurodegenerative diseases such as Parkinson's disease, Huntington's disease or schizophrenia using transgenic animals in which D 2 S and DL are no longer expressed, including
* l'administration à un mammifère non humain transgénique ou à des cellules de mammifères non humains transgéniques contenant une construction choisie parmi:* administration to a transgenic non-human mammal or to cells of transgenic non-human mammals containing a construct chosen from:
- le gène de D2 dans lequel un fragment contenant l'exon 2 est remplacé par un gène marqueur sous le contrôle d'un promoteur approprié, notamment une cassette contenant le gène neo sous le contrôle du promoteur PGKI, et en particulier le gène de D2 dans lequel le fragment génomique de 0,9 Kb Ncol, contenant l'exon 2 est remplacé par la cassette PGK-neo du composé à tester, * et la détermination de l'amélioration de l'état neurodégénératif.the gene for D 2 in which a fragment containing exon 2 is replaced by a marker gene under the control of an appropriate promoter, in particular a cassette containing the neo gene under the control of the PGKI promoter, and in particular the gene for D 2 in which the 0.9 Kb Ncol genomic fragment containing exon 2 is replaced by the PGK-neo cassette of the compound to be tested, * and the determination of the improvement of the neurodegenerative state.
S 'agissant de l'amélioration de l'état neurodégénératif chez les Parkinsoniens, on s'attend à une diminution du tremblement de repos et aussi à une amélioration de la posture et des mouvements volontaires.Regarding the improvement of the neurodegenerative state in Parkinson's, we expect a decrease in rest tremor and also an improvement in posture and voluntary movements.
On attend principalement que ces souris puissent représenter un modèle d'étude pour les maladies comme la maladie de Parkinson ou la schizophrénie.It is mainly expected that these mice can represent a study model for diseases such as Parkinson's disease or schizophrenia.
Il n'est pas exclu que des drogues qui pourraient être testées chez les animaux transgéniques de l'invention puissent aussi améliorer des maladies qui n'ont pas d'anomalies du récepteur D2 à leur base.It is not excluded that drugs which could be tested in the transgenic animals of the invention can also improve diseases which do not have abnormalities of the D 2 receptor at their base.
Un transgène est défini comme un fragment d'ADN qui est inséré de façon artificielle (c'est-à-dire autrement que par reproduction sexuelle) dans une cellule et qui devient partie intégrante au génome de l'animal qui se développe à partir de cette cellule, ou qui est modifié par rapport à l'ADN normalement présent dans la cellule. Un tel transgène peut être un gène homologue à un gène naturel de l'animal transgénique mais qui est inséré dans le génome de l'animal à une position qui diffère de celle de l'homologue naturel ou bien un tel transgène peut correspondre au gène homologue modifié, notamment par une délétion d'un fragment d'ADN dudit gène naturel.
L' invention concerne également un procédé de préparation d'un animal transgénique, portant le susdit transgène, qui peut être élevé de façon stable pour donner des descendants comportant des cellules qui contiennent le gène de façon stable. Ce procédé peut comprendre: - le prélèvement d'un ovocyte fécondé à partir d'un premier animal femelle,A transgene is defined as a fragment of DNA which is inserted artificially (i.e. other than by sexual reproduction) in a cell and which becomes part of the genome of the animal which develops from that cell, or that is modified from the DNA normally present in the cell. Such a transgene may be a gene homologous to a natural gene of the transgenic animal but which is inserted into the genome of the animal at a position which differs from that of the natural counterpart, or else such a transgene may correspond to the homologous gene. modified, in particular by deletion of a DNA fragment of said natural gene. The invention also relates to a process for the preparation of a transgenic animal, carrying the aforementioned transgene, which can be raised stably to give offspring comprising cells which contain the gene stably. This process can include: - the removal of a fertilized oocyte from a first female animal,
- le transfert d'un transgène dans l' ovocyte fécondé,- the transfer of a transgene into the fertilized oocyte,
- le transfert de 1 Ovocyte fécondé contenant le transgène dans l'oviducte d'un deuxième animal femelle de la même espèce que le susdit premier animal,- the transfer of 1 fertilized oocyte containing the transgene into the oviduct of a second female animal of the same species as the above first animal,
- le maintien du deuxième animal femelle dans un état tel que:- keeping the second female animal in a state such that:
- ledit animal femelle commence une gestation par rapport à l'embryon dérivé de l' ovocyte fécondé contenant le transgène,- said female animal begins gestation with respect to the embryo derived from the fertilized oocyte containing the transgene,
- l'embryon se développe en animal transgénique, - l'animal transgénique naît du deuxième animal femelle.- the embryo develops into a transgenic animal, - the transgenic animal is born from the second female animal.
Description des figures:Description of the figures:
- La figure 1A représente la construction PRLD L.- Figure 1A shows the construction PRLD L.
- La figure 1B représente la construction PRLD S.- Figure 1B shows the construction PRLD S.
- la figure 1C représente le fragment BamHI-BglII qui sert de sonde pour déterminer s'il y a eu intégration du transgène. Dans ce qui précède et ce qui suit, on désigne par "construction ou transgène PRLD2L", la construction transgénique ou le gène contenant ladite construction, permettant la surexpression de D L, et par "construction ou transgène PRLD S", la construction transgénique ou le gène contenant ladite construction, permettant la surexpression de D2S. On désigne par "souris transgéniques PRLD L ou PRLD2S", les souris contenant la construction transgénique PRLD2L ou PRLD2S.- Figure 1C shows the BamHI-BglII fragment which serves as a probe to determine whether there has been integration of the transgene. In what precedes and what follows, one designates by “construction or transgene PRLD 2 L”, the transgenic construction or the gene containing said construction, allowing the overexpression of DL, and by “construction or transgene PRLD S”, the transgenic construction or the gene containing said construct, allowing the overexpression of D 2 S. The term “PRLD L or PRLD 2 S transgenic mice” denotes mice containing the transgenic construct PRLD 2 L or PRLD 2 S.
- La figure 2 représente l'analyse par PCR de souris comportant à la fois la construction PRLD2L et PRLD2S. Afin d'analyser les souris provenant de l'accouplement entre les lignées trasngéniques PRLD L et PRLD S, on prépare l'ADN à partir d'un petit fragment de queue de ces animaux. La réaction de polymérisation en chaîne (PCR) est faite en présence de deux amorces, l'une correspondant aux
séquences communes des ARNm de D2L et D2S comprises, par exemple, entre les nucléotides 830 à 850 des ADNc de souris et l'autre correspondant, par exemple, aux nucléotides compris entre les positions 4457 et 4477 des séquences du virus SV40 qui sont présentes dans le fragment qui a servi pour la construction du transgène. L'utilisation de ces amorces conduit à l'obtention de deux fragments: l'un de 1722 paires de bases correspondant au transgène D2L et l'autre de 1635 paires de bases correspondant au transgène D S. L'intérêt de cette méthode est que seules les séquences correspondant aux transgènes sont amplifiées, tandis que le gène endogène ne l'est pas car il ne contient pas les séquences de SV40. La présence simultanée des deux fragments montre que l'animal en question a les deux transgènes D2L et D2S intégrés dans son génome.FIG. 2 represents the PCR analysis of mice comprising both the PRLD 2 L and PRLD 2 S construct. In order to analyze the mice originating from the coupling between the trasngene lines PRLD L and PRLD S, l DNA from a small tail fragment from these animals. The polymerase chain reaction (PCR) is carried out in the presence of two primers, one corresponding to the common sequences of D 2 L and D 2 S mRNAs included, for example, between nucleotides 830 to 850 of mouse cDNAs and the other corresponding, for example, to nucleotides between positions 4457 and 4477 of SV40 virus sequences which are present in the fragment which served for the construction of the transgene. The use of these primers leads to the production of two fragments: one of 1722 base pairs corresponding to the D 2 L transgene and the other of 1635 base pairs corresponding to the D S transgene. The advantage of this method is that only the sequences corresponding to the transgenes are amplified, while the endogenous gene is not because it does not contain the SV40 sequences. The simultaneous presence of the two fragments shows that the animal in question has the two transgenes D 2 L and D 2 S integrated into its genome.
- La figure 3 représente la recombinaison homologue conduisant à la préparation de souris transgéniques n'exprimant plus ni D2S ni D2L.- Figure 3 shows the homologous recombination leading to the preparation of transgenic mice no longer expressing either D 2 S or D 2 L.
- La figure 4 représente la recombinaison homologue conduisant à la préparation de souris transgéniques n'exprimant plus D2L.- Figure 4 shows the homologous recombination leading to the preparation of transgenic mice no longer expressing D 2 L.
- La figure 5 représente la séquence amino acide et nucléotidique de- Figure 5 shows the amino acid and nucleotide sequence of
D2L.D 2 L.
La figure 6 représente la séquence amino acide et nucléotidique deFIG. 6 represents the amino acid and nucleotide sequence of
D2S.D 2 S.
- La figure 7 représente l'analyse de l'ARN provenant d'hypophyses de souris transgéniques PRLD2L et PRLD2S comparé à des souris sauvages, et montre la surexpression des isoformes D2L ou D2S.FIG. 7 represents the analysis of the RNA originating from the pituitaries of transgenic mice PRLD 2 L and PRLD 2 S compared to wild mice, and shows the overexpression of the isoforms D 2 L or D 2 S.
On effectue la protection par la digestion avec l'enzyme RNAse provenant d'hypophyses de souris transgéniques PRLD2L, PRLD2S et de leur contrôles (C) (frère non transgénique, de la même portée), de la sonde à ARN hybride à l'ARN correspondant à l'ARN messager du gène D2, cette sonde étant décrite dans le texte (fragment AccI dans pBluescript, plasmide linéarisé par Bsu36I). Les flèches indiquent les bandes correspondant à la taille attendue pour D2L (202 nucléotides) et D2S (130 nucléotides).Protection is carried out by digestion with the enzyme RNAse from the pituitaries of transgenic mice PRLD 2 L, PRLD 2 S and their controls (C) (non-transgenic sibling, of the same litter), from the hybrid RNA probe to the RNA corresponding to the messenger RNA of the D 2 gene, this probe being described in the text (AccI fragment in pBluescript, plasmid linearized by Bsu36I). The arrows indicate the bands corresponding to the expected size for D 2 L (202 nucleotides) and D 2 S (130 nucleotides).
On peut observer dans la ligne correspondant à l'ARN de souris contrôle que la forme D2L est bien plus exprimée dans l'hypophyse de l'animal contrôle que la forme D2S. Le contraste de la photographie augmente encore
plus cette différence, de telle façon que l'on ne voit plus que la forme D2L dans le contrôle. De plus longues expositions du gel permettent de visualiser un signal aussi pour D2S dans le contrôle. La quantification de la surexpression de D2L dans la ligne correspondant à l'hypophyse de souris PRLD L, donne une augmentation de 7 fois de l'expression de D2L dans les souris transgéniques par rapport aux souris contrôle. En ce qui concerne la ligne correspondant aux souris PRLD2S, la quantification nous donne une augmentation de 10 fois de D2S dans ces souris transgéniques par rapport à leur contrôle. II faut noter que la surexpression des transgènes PRLD2L ou PRLD S se rajoute à l'expression du gène endogène.It can be observed in the line corresponding to the control mouse RNA that the form D 2 L is much more expressed in the pituitary gland of the control animal than the form D 2 S. The contrast of the photograph increases further plus this difference, so that we only see the form D 2 L in the control. Longer exposures of the gel allow visualization of a signal also for D 2 S in the control. The quantification of the overexpression of D 2 L in the line corresponding to the pituitary gland of PRLD L mice gives a 7-fold increase in the expression of D 2 L in transgenic mice compared to the control mice. With regard to the line corresponding to PRLD 2 S mice, the quantification gives us a 10-fold increase in D 2 S in these transgenic mice compared to their control. It should be noted that the overexpression of the PRLD 2 L or PRLD S transgenes is added to the expression of the endogenous gene.
La figure 8 représente la protection à l'enzyme RNAse de l'ARN provenant de tissus de souris (souris normales ou sauvages, hétérozygotes ou homozygotes) dans lesquelles on a éliminé la forme D2L du récepteur.FIG. 8 represents the protection with the RNAse enzyme of RNA originating from tissues of mice (normal or wild mice, heterozygous or homozygous) in which the D 2 L form of the receptor has been eliminated.
Les ARN de striatum, substantia nigra, cortex et cerebellum de souris normales (+/+), hétérozygotes (+/-) et homozygotes (-/-) ont été hybrides avec la sonde correspondant au plasmide contenant le fragment AccI coupé par Bsu36I et transcrit par l'enzyme T3. On observe clairement la disparition de la bande correspondant à la forme D2L, chez les souris homozygotes. Sur cette photographie, on n'observe des bandes correspondant au transcrit D2 que dans le striatum, et la substantia nigra comme attendu, les autres régions du cerveau utilisées (cortex, cerebellum) servant de contrôle négatif.RNAs from striatum, substantia nigra, cortex and cerebellum of normal (+ / +), heterozygous (+/-) and homozygous (- / -) mice were hybridized with the probe corresponding to the plasmid containing the AccI fragment cut by Bsu36I and transcribed by the T3 enzyme. The disappearance of the band corresponding to the D 2 L form is clearly observed in homozygous mice. In this photograph, bands corresponding to the D 2 transcript are observed only in the striatum, and the substantia nigra as expected, the other regions of the brain used (cortex, cerebellum) serving as negative control.
La figure 9 représente l'absence d'expression de D2 dans les souris transgéniques obtenues par recombinaison homologue.FIG. 9 represents the absence of expression of D 2 in the transgenic mice obtained by homologous recombination.
A) Analyse par la technique de Northern des ARN provenant du striatum de souris normales (+/+), hétérozygotes (+/-) et homozygotes (-/-) pour l'élimination de l'expression du gène du récepteur dopaminergique D2. La sonde utilisée correspond au fragment Smal-PvuII décrit dans le texte.A) Northern analysis of RNA from the striatum of normal (+ / +), heterozygous (+/-) and homozygous (- / -) mice for the elimination of expression of the D 2 dopaminergic receptor gene . The probe used corresponds to the Smal-PvuII fragment described in the text.
B) Analyse par la méthode de Scatchard d'une courbe de saturation obtenue par liaison de 3H-spipérone à des membranes de striatum de souris. Cette analyse montre que dans les souris hétérozygotes, le nombre de sites D2 est diminué par rapport à la souris sauvage, et est complètement absent dans les souris homozygotes qui n'ont plus les récepteurs D2.B) Analysis by the Scatchard method of a saturation curve obtained by binding of 3H-spiperone to membranes of mouse striatum. This analysis shows that in heterozygous mice, the number of D 2 sites is reduced compared to the wild mouse, and is completely absent in homozygous mice which no longer have the D 2 receptors.
Méthode: les striata provenant de souris sauvages (carré blanc), hétérozygotes (triangle noir), et homozygotes (rond blanc) sont homogénéisés avec un homogénéisateur Ultra-Turrax dans 1 ml de Tris-HCl, 10 mM, pH
7,5 froid, contenant 5 mM EDTA. Les membranes sont centrifugées à 1000 g pendant 10 min, le surnageant est récupéré puis centrifugé à 45 000 g pendant 40 min à 4°C. Le culot est lavé puis repris dans 50 mM Tris-HCl pH 7,7. Pour l'analyse du récepteur D2, 15 μg de membranes sont incubés dans une solution de Tris-HCl 50 mM, pH 7,7, contenant 120 mM NaCl, 5 mM KC1, 2 mM CaCl2, 1 mM MgCl2, 0,1% acide ascorbique et 3H-spipérone (10-600 pM; activité spécifique 114 Ci/mmol; Amersham) à 37°C pendant 60 min. La liaison non spécifique est déterminée en présence de 1 μM (+)- butaclamol (RBI, Natick, MA).Method: the striata from wild mice (white square), heterozygotes (black triangle), and homozygotes (white circle) are homogenized with an Ultra-Turrax homogenizer in 1 ml of Tris-HCl, 10 mM, pH 7.5 cold, containing 5 mM EDTA. The membranes are centrifuged at 1000 g for 10 min, the supernatant is collected and then centrifuged at 45,000 g for 40 min at 4 ° C. The pellet is washed and then taken up in 50 mM Tris-HCl pH 7.7. For the analysis of the D 2 receptor, 15 μg of membranes are incubated in a 50 mM Tris-HCl solution, pH 7.7, containing 120 mM NaCl, 5 mM KC1, 2 mM CaCl 2 , 1 mM MgCl 2 , 0 , 1% ascorbic acid and 3H-spiperone (10-600 pM; specific activity 114 Ci / mmol; Amersham) at 37 ° C for 60 min. Nonspecific binding is determined in the presence of 1 μM (+) - butaclamol (RBI, Natick, MA).
Le ligand radioactif lié aux membranes est récupéré par filtration rapide à travers des filtres en fibres de verre Whatmann GF/B. Les filtres sont lavés trois fois avec 5 ml de Tris-HCl 50 mM, pH 7,7 froid.The radioactive ligand bound to the membranes is recovered by rapid filtration through Whatmann GF / B glass fiber filters. The filters are washed three times with 5 ml of 50 mM Tris-HCl, pH 7.7 cold.
Les données sont analysées avec le programme LIGAND (BIOSOFT). En abscisse, on a indiqué la quantité de 3H-spipérone liée au récepteurThe data is analyzed with the LIGAND program (BIOSOFT). On the abscissa, the amount of 3H-spiperone linked to the receptor is indicated.
(f mol/mg de protéine).(f mol / mg protein).
En ordonnées, on a indiqué le rapport 3H-spipérone liée/3H-spipérone libre.On the ordinate, the ratio 3H-bound spiperone / 3H-free spiperone was indicated.
Exemples;Examples;
Exemple 1: Préparation de souris transgéniques surexprimant D L ou D2S.EXAMPLE 1 Preparation of Transgenic Mice Overexpressing DL or D 2 S.
Afin de générer des souris transgéniques surexprimant l'une des deux formes des récepteurs dopaminergiques D2 sur des cellules lactotropes pituitaires (de l'hypophyse) de souris, on a utilisé le promoteur du gène de la prolactine.In order to generate transgenic mice overexpressing one of the two forms of the dopaminergic D 2 receptors on mouse pituitary (pituitary) lactotropic cells, the prolactin gene promoter was used.
Un fragment de 3,03 Kb BamHI-HindIII (- 3000/ +33) du promoteur du gène de la prolactine (Borrelli, E., Sa chenko, P. et Evans, R. (1992) Proc.A 3.03 Kb BamHI-HindIII fragment (- 3000 / +33) of the prolactin gene promoter (Borrelli, E., Sa chenko, P. and Evans, R. (1992) Proc.
Natl. Acad. Sci. USA, 89: 2764-2768) a été fusionné avec le fragment de 2,261 Kb d'ADNc Smal-EcoRV de D2L (Montmayeur, J.-P., Bausero, P., Amlaiky, N., Maroteaux, L., Hen, R. et Borrelli, E. (1991) Febs lett., 278: 239-243) ou avec le fragment de 2,174 Kb d'ADNc de D2S (Montmayeur, J.-P., Bausero, P., Amlaiky, N., Maroteaux, L., Hen, R. et Borrelli, E.Natl. Acad. Sci. USA, 89: 2764-2768) was fused with the 2.261 Kb fragment of Smal-EcoRV cDNA of D 2 L (Montmayeur, J.-P., Bausero, P., Amlaiky, N., Maroteaux, L. , Hen, R. and Borrelli, E. (1991) Febs lett., 278: 239-243) or with the 2.174 Kb fragment of D 2 S cDNA (Montmayeur, J.-P., Bausero, P. , Amlaiky, N., Maroteaux, L., Hen, R. and Borrelli, E.
(1991) Febs lett., 278: 239-243); le site de polyadénylation et les séquences d'intron du petit antigène T du virus SV40 contenues dans le fragment de 847 paires de base BamHI-BglII (Subramani, S. et Southern, P. J. (1983) Anal.
Biochem., 135: 1-15) ont été fusionnées en aval des ADNc du récepteur D2, générant les constructions de fusion PRLD2L et PRLD2S. Ces fragments ont été excisés aux sites Not I flanquant les constructions et injectés dans le pronucleus mâle d'ovocytes fertilisés de souris C57 Black 6/SJL (provenant de Jackson Laboratory, Barharbor, Maine, 04609, USA).(1991) Febs lett., 278: 239-243); the polyadenylation site and the intron sequences of the small antigen T of the virus SV40 contained in the fragment of 847 base pairs BamHI-BglII (Subramani, S. and Southern, PJ (1983) Anal. Biochem., 135: 1-15) were fused downstream of the D 2 receptor cDNAs, generating the PRLD 2 L and PRLD 2 S fusion constructs. These fragments were excised at the Not I sites flanking the constructs and injected into the male pronucleus of fertilized oocytes from C57 Black 6 / SJL mice (from Jackson Laboratory, Barharbor, Maine, 04609, USA).
Les souris qui naissent de ces injections sont testées pour la présence de la construction transgénique en analysant l'ADN génomique obtenu à partir d'un petit échantillon de la queue. L'ADN obtenu est testé par analyse de transfert d'ADN (Southern blot) digéré par PstI, mis en contact avec une sonde constituée d'un fragment de SV40 BamHI-BglII marqué au 32p; seules les souris transgéniques sont révélées de cette façon, étant donné que l'ADN des souris sauvages (c'est-à-dire non transgéniques) ne s 'hybride pas avec cette sonde.Mice born from these injections are tested for the presence of the transgenic construct by analyzing genomic DNA obtained from a small sample of the tail. The DNA obtained is tested by DNA transfer analysis (Southern blot) digested with PstI, brought into contact with a probe consisting of a fragment of SV40 BamHI-BglII labeled with 32p ; only transgenic mice are revealed in this way, since the DNA of wild (i.e. non-transgenic) mice does not hybridize with this probe.
Des lignées de souris sont obtenues en croisant des souris mâles ou femelles transgéniques avec des souris C57 Black 6 provenant de JacksonMouse lines are obtained by crossing male or female transgenic mice with C57 Black 6 mice from Jackson
Laboratory. Les souris hétérozygotes sont testées pour l'expression du transgène par analyse de l'ARNm pituitaire par la méthode de protection à la RNAse, en utilisant une sonde spécifique (fragment AccI dans pBluescript linéarisé par Bsu36I) du récepteur D2 et par analyse de transfert d'ARN (Northern blot), en utilisant le fragment SV40 BamHI-BglII marqué au 32p.Laboratory. Heterozygous mice are tested for transgene expression by analysis of pituitary mRNA by the RNAse protection method, using a specific probe (AccI fragment in pBluescript linearized by Bsu36I) of the D 2 receptor and by transfer analysis RNA (Northern blot), using the SV40 BamHI-BglII fragment labeled with 32p.
Deux lignées surexprimant les constructions PRLD2L sont obtenues; une lignée est obtenue pour la construction PRLD2S.Two lines overexpressing the PRLD 2 L constructs are obtained; a line is obtained for the construction PRLD 2 S.
Exemple 2: Préparation de souris transgéniques surexprimant D2L et D2S.Example 2 Preparation of Transgenic Mice Overexpressing D 2 L and D 2 S.
Afin d'obtenir des souris surexprimant les deux isoformes des récepteurs D2, on croise des femelles PRLD2L hétérozygotes avec des mâles PRLD2S. Pour tester les souris positives vis à vis de ces deux transgènes, on utilise une technique d'amplification en chaîne par polymérase (PCR). L'ADN génomique de la queue de souris est amplifié en utilisant des oligonucléotides synthétiques appartenant au fragment ajouté SV40 et par exemple s 'étendant du nucléotide en position 4457 au nucléotide en position 4477 du génome du virus SV40, et à l'ADNc des récepteurs de la dopamine. Des amorces oligonucléotidiques sont choisies dans la région des ADNc des récepteurs D en amont de la présente insertion dans D2L (par exemple de la position 830 à la position 850 de l'ADNc de souris). Deux fragments de longueur différente sont obtenus, mettant en évidence le caractère doublement transgénique (voir figure 2).
Exemple 3: Préparation de souris transgéniques n'exprimant plus ni D S ni D L (recombinaison homologue).To obtain mice overexpressing both isoforms of D 2 receptors, are crossed female LTIP 2 L heterozygous males with LTIP 2 S. To test positive mice with respect to these two transgenes, an amplification technique is used polymerase chain reaction (PCR). The genomic DNA of the mouse tail is amplified using synthetic oligonucleotides belonging to the added fragment SV40 and for example extending from the nucleotide at position 4457 to the nucleotide at position 4477 of the genome of the SV40 virus, and to the cDNA of the receptors dopamine. Oligonucleotide primers are chosen from the D receptor cDNA region upstream of the present insertion into D 2 L (for example from position 830 to position 850 of mouse cDNA). Two fragments of different length are obtained, highlighting the doubly transgenic character (see FIG. 2). Example 3: Preparation of transgenic mice no longer expressing DS or DL (homologous recombination).
Pour supprimer l'expression totale du récepteur D2, de l'ADN génomique clone correspondant au site du récepteur D2 est isolé à partir d'une banque génomique de souris ES/EMBL3: (Sambrook J., Fritsch, E. F., Maniatis, T., Molecular Cloning, 2ème édition, Ed. Cold Spring Harbor Laboratory Press, 1989, vol. 2, chapitre 9). Les cellules ES proviennent de la souris souche 129 de couleur agouti (Jackson Laboratory). Un fragment génomique de souris de 6,5 Kb Kpnl-Sall contenant l'exon 2 (Karen L.To suppress the total expression of the D 2 receptor, cloned genomic DNA corresponding to the D 2 receptor site is isolated from a genomic library of ES / EMBL3 mice: (Sambrook J., Fritsch, EF, Maniatis, T., Molecular Cloning, 2nd edition, Ed. Cold Spring Harbor Laboratory Press, 1989, vol. 2, chapter 9). The ES cells come from the mouse strain 129 of agouti color (Jackson Laboratory). A 6.5 kb Kpnl-Sall mouse genomic fragment containing exon 2 (Karen L.
O'Malley et al. (1990). Organization and Expression of the Rat D2A Receptor Gène: Identification of Alternative Transcripts and a Variant Donor Splice Site. Biochemistry, 29: 1367-1371) du gène D2 est souscloné dans pBluescript. A partir de ce plasmide, le fragment Ncol de 0,9 Kb contenant l'exon 2 est éliminé et remplacé par un segment de 1 ,4 Kb du gène de résistance à la néomycine sous le contrôle du promoteur de la phosphoglycérate kinase-1 (PGK-1) dans la direction transcriptionnelle opposée. Le vecteur ciblant est linéarisé aux sites uniques Kpnl ou Sali présent dans le site de clonage multiple avant électroporation des cellules PI ES, qui peuvent être obtenues auprès de l'IGBMC (Institut de Génétique et de BiologieO'Malley et al. (1990). Organization and Expression of the Rat D2A Receptor Gene: Identification of Alternative Transcripts and a Variant Donor Splice Site. Biochemistry, 29: 1367-1371) of the D 2 gene is subcloned in pBluescript. From this plasmid, the 0.9 kb Ncol fragment containing exon 2 is eliminated and replaced by a 1.4 kb segment of the neomycin resistance gene under the control of the phosphoglycerate kinase-1 promoter ( PGK-1) in the opposite transcriptional direction. The targeting vector is linearized at the unique Kpnl or Sali sites present in the multiple cloning site before electroporation of the PI ES cells, which can be obtained from the IGBMC (Institute of Genetics and Biology
Moléculaire et Cellulaire, Illkirch, France).Molecular and Cellular, Illkirch, France).
Pour l' électroporation, 1.10^ cellules PI ES sont mélangées avec 10 μg de construction linéarisée dans 0,5 ml de DMEM (DMEM = Dulbecco modified Eagle's médium) plus 15% de FCS (FCS = sérum de veau foetal) dans une cuvette pour l' électroporation de 0,4 cm et électroporée à 400 VoltFor electroporation, 1.10 ^ PI ES cells are mixed with 10 μg of linearized construction in 0.5 ml of DMEM (DMEM = Dulbecco modified Eagle's medium) plus 15% of FCS (FCS = fetal calf serum) in a cuvette. electroporation of 0.4 cm and electroporated at 400 Volt
(1000 V/cm), à 125 μFarads en utilisant un appareil d 'électroporation commercialisé sous le nom Gène Pulsar (Bio-Rad). Les cellules sont ensuite transférées dans un milieu non sélectif (Dulbecco) pendant 2 jours et sélectionnées dans 150 μg/ml de gentamycine (G418) pendant 10 jours. Les clones ES résistants sont isolés et mis dans des micropuits contenant une couche nourricière cellulaire (fibroblastes irradiés) et ensuite multipliés pour l'analyse d'ADN par la technique de transfert d'ADN (Southern blot). La digestion d'ADN avec Spel a donné un fragment de 12,5 Kb à partir de type sauvage et de 9,5 Kb à partir de l'allèle ciblée, lorsqu'elle est hy bridée avec une sonde externe, à la partie de D2 impliquée dans la recombinaison homologue, et notamment la sonde comprenant ou constituée par le fragment situé entre les exons 3 et 4 de D2, coupé par l'enzyme PstI (figure 3). Les cellules ES portant le gène D généré par recombinaison homologue sont
injectées dans des embryons de C57BL/6 provenant de Jackson Laboratory à l'état de blastocytes, en utilisant des techniques standard (Bradley, A. (1987) Production and analysis of chimaeric mice. In teratocarcinomas and Embryonic Stem Cells: A practical Approach, Robertson, E. J., éd. (Oxford: IRL Press Limited)). Les descendants chimères sont identifiés grâce à la couleur du poil correspondant au phénotype chimérique (couleur grise et noire) et sont croisés avec des souris C57BL/6. La transmission de la lignée germinale de l'allele mutante est déterminée par transfert d'ADN (Southern blot) de l'ADN prélevée dans la queue isolée de la progénie "agouti" par digestion avec Spel. Des souris homozygotes vis à vis de la mutation D sont obtenues en croisant des souris mutantes hétérozygotes.(1000 V / cm), at 125 μFarads using an electroporation device sold under the name Gene Pulsar (Bio-Rad). The cells are then transferred to a non-selective medium (Dulbecco) for 2 days and selected in 150 μg / ml of gentamycin (G418) for 10 days. The resistant ES clones are isolated and placed in microwells containing a cellular nourishing layer (irradiated fibroblasts) and then multiplied for DNA analysis by the DNA transfer technique (Southern blot). Digestion of DNA with Spel gave a fragment of 12.5 Kb from wild type and 9.5 Kb from the targeted allele, when hybridized with an external probe, to the part of D 2 involved in homologous recombination, and in particular the probe comprising or consisting of the fragment located between exons 3 and 4 of D 2 , cut by the enzyme PstI (FIG. 3). ES cells carrying the D gene generated by homologous recombination are injected into blastocyte C57BL / 6 embryos from Jackson Laboratory using standard techniques (Bradley, A. (1987) Production and analysis of chimaeric mice. In teratocarcinomas and Embryonic Stem Cells: A practical Approach, Robertson, EJ, ed. (Oxford: IRL Press Limited)). The chimeric descendants are identified by the color of the hair corresponding to the chimeric phenotype (gray and black color) and are crossed with C57BL / 6 mice. The transmission of the germ line of the mutant allele is determined by DNA transfer (Southern blot) from the DNA taken from the tail isolated from the "agouti" progeny by digestion with Spel. Mice homozygous towards the D mutation are obtained by crossing mutant heterozygous mice.
Exemple 4: Préparation de souris n'exprimant plus D L par recombinaison homologue.Example 4: Preparation of mice no longer expressing D L by homologous recombination.
Pour obtenir les souris transgéniques n'exprimant plus D2L, un fragment génomique de 4,8 Kb EcoRI et contenant l'exon 3 et l'exon 4 est sous-cloné dans pBluescript, un fragment de 1,5 Kb BglII entourant l'exon 6 est supprimé et remplacé par le gène cassette PGK-neo. A ce plasmide, un autre fragment génomique de 5,8 Kb EcoRI allant de l'exon 5 à l'exon 7 est fusionné pour fournir un fragment de plus grande homologie. Pour éliminer l'intégration au hasard, le gène viral de la thymidine kinase (TK) de l'herpès simple sous le contrôle du promoteur PGK, est inclus à l'extrémité 3' du vecteur. Le vecteur ciblant est linéarisé au site Notl unique dans le site de clonage multiple avant électroporation des cellules ES D3 (Dr. Kemler Institut Max Planck, Fribourg,To obtain transgenic mice no longer expressing D 2 L, a 4.8 Kb EcoRI genomic fragment containing exon 3 and exon 4 is subcloned into pBluescript, a 1.5 Kb BglII fragment surrounding l exon 6 is deleted and replaced by the PGK-neo cassette gene. To this plasmid, another 5.8 Kb EcoRI genomic fragment going from exon 5 to exon 7 is fused to provide a fragment of greater homology. To eliminate random integration, the viral gene for simple herpes thymidine kinase (TK) under the control of the PGK promoter is included at the 3 'end of the vector. The targeting vector is linearized at the unique NotI site in the multiple cloning site before electroporation of ES D3 cells (Dr. Kemler Institut Max Planck, Friborg,
Allemagne). Les cellules ES D3 électroporées sont sélectionnées dans 150 μg/ml de gentamycine (G418) + gancyclovir 2 μM pendant 10 jours. Les clones ciblés sont identifiés par transfert d'ADN génomique (Southern blot) coupé avec Bgll et hybrides à une sonde externe provenant d'un fragment de D2 entourant l'exon 8 et digéré par EcoRI-BglI (figure 4). La digestion d'ADN avec Bgll a donné un fragment de 6,8 Kb à partir de type sauvage et 4,5 Kb à partir d'allèle ciblée. Le procédé pour générer des souris mutantes ne possédant plus l'isoforme D2L est effectué selon ce qui a été décrit à partir des souris n'exprimant plus ni le récepteur D2S ni le récepteur D2L.
Exemple 5 : caractérisation des souris n'exprimant plus le récepteur D , (souris D2 knock-out) ou n'exprimant plus D2L.Germany). Electroporated ES D3 cells are selected from 150 μg / ml gentamycin (G418) + 2 μM gancyclovir for 10 days. The targeted clones are identified by transfer of genomic DNA (Southern blot) cut with Bgl1 and hybridized to an external probe originating from a fragment of D 2 surrounding exon 8 and digested with EcoRI-BglI (FIG. 4). Digestion of DNA with Bgl1 gave a fragment of 6.8 Kb from wild type and 4.5 Kb from targeted allele. The process for generating mutant mice no longer having the D 2 L isoform is carried out according to what has been described from the mice no longer expressing either the D 2 S receptor or the D 2 L receptor. Example 5: Characterization of the mice no longer expressing the D receptor (D 2 knockout mice) or no longer expressing D 2 L.
Les souris D2 knock-out présentent un phénotype Parkinsonien, mouvements très réduits, mauvaise coordination et akinésie. Le phénotype de ces souris au niveau de la locomotion démontre que le récepteur D2, et non les autres récepteurs dopaminergiques (tels que Di , D3, D4 et D5), est le récepteur dopaminergique responsable de cette fonction. La similarité du phénotype entre les souris D - / - (n'exprimant que le récepteur D2) et la maladie de Parkinson suggère que les récepteurs D2 sont les plus affectés des récepteurs dopaminergiques par le manque de dopamine chez les malades. Cette observation valide ces souris comme modèle d'étude de la maladie de Parkinson. On peut étudier quels sont les autres circuits neuronaux affectés par le manque d'activité des récepteurs D2 et essayer sur ces animaux des drogues capables de réduire ces effets. A présent, la maladie de Parkinson n'est traitée que par prise de L-DOPA, le précurseur métabolique de la dopamine. On a aussi trouvé que dans les animaux de l'invention, il y a augmentation de la synthèse de l'enzyme G AD (acide glutamique décarboxylase) déterminée au niveau de l'ARNm par hybridation in situ et peut être donc du neurotransmetteur GABA (acide γ-aminobutyrique).D2 knockout mice exhibit a Parkinsonian phenotype, very reduced movement, poor coordination and akinesia. The locomotion phenotype of these mice demonstrates that the D 2 receptor, and not the other dopaminergic receptors (such as Di, D3, D4 and D5), is the dopaminergic receptor responsible for this function. The similarity of the phenotype between D - / - mice (expressing only the D 2 receptor) and Parkinson's disease suggests that D 2 receptors are the most affected of dopaminergic receptors by the lack of dopamine in patients. This observation validates these mice as a model for studying Parkinson's disease. We can study what other neural circuits are affected by the lack of D 2 receptor activity and try drugs capable of reducing these effects on these animals. Parkinson's disease is now treated only by taking L-DOPA, the metabolic precursor of dopamine. It has also been found that in the animals of the invention, there is an increase in the synthesis of the enzyme G AD (glutamic acid decarboxylase) determined at the level of the mRNA by in situ hybridization and therefore may be of the neurotransmitter GABA ( γ-aminobutyric acid).
Plus récemment on a analysé par voie électrophysiologique les neurones du striatum des souris D2 - / - et on a découvert que la plasticité synaptique de ces neurones est modifiée, et peut être responsable du phénotype locomoteur. S 'agissant de la plasticité synaptique (Calabresi P. et al. Trends Neurosci. 19, 19-24, 1996), la stimulation tétanique de fibres corticostriatales est connue pour produire une dépression synaptique à long terme (LTD) dans les souris de type sauvage. De façon surprenante, la réponse inverse, la potentialisation à long terme (LTP) est observée à partir de souris n'exprimant pas D2. La potentialisation à long terme (LTP) à la différence de la dépression synaptique à long terme (LTD) est bloquée par un antagoniste du récepteur auMore recently, the neurons of the striatum of D 2 - / - mice have been analyzed electrophysiologically and it has been discovered that the synaptic plasticity of these neurons is modified, and may be responsible for the locomotor phenotype. As regards synaptic plasticity (Calabresi P. et al. Trends Neurosci. 19, 19-24, 1996), tetanus stimulation of corticostriatal fibers is known to produce long-term synaptic depression (LTD) in type mice wild. Surprisingly, the opposite response, long-term potentiation (LTP) is observed from mice not expressing D 2 . Long-term potentiation (LTP) unlike long-term synaptic depression (LTD) is blocked by a receptor antagonist
N-méthyl-d-aspartate .N-methyl-d-aspartate.
On a aussi étudié si le récepteur dopaminergique D2 intervient dans la dépendance aux drogues telles que la morphine. On a trouvé que les souris D - / - ne deviennent plus dépendantes de la morphine après des traitements prolongés avec cette drogue. Ces animaux, même s'ils n'ont plus de dépendance à la morphine, présentent toujours le syndrome de sevrage, quand ils sont traités avec un antagoniste des récepteurs aux opiacés. Ces souris représentent à ce niveau un modèle d'étude de la dépendance aux opiacés et
encore plus un modèle pour étudier des composés agissant uniquement au niveau du sevrage des drogues.We have also studied whether the dopaminergic receptor D 2 is involved in dependence on drugs such as morphine. It has been found that D - / - mice no longer become dependent on morphine after prolonged treatments with this drug. These animals, even if they are no longer addicted to morphine, still exhibit withdrawal syndrome when they are treated with an opioid receptor antagonist. At this level, these mice represent a model for studying opiate dependence and even more a model for studying compounds acting only at the level of drug withdrawal.
Les souris de l'invention peuvent servir d'outil pour valider des composés visant des autres récepteurs dopaminergiques de la sous-famille D2, tels que D3 et D4, ou de la sous-famille Di, tels que O et D5 avec une spécificité pour D2 uniquement et non pour D^ et D2.The mice of the invention can serve as a tool for validating compounds targeting other dopaminergic receptors of the D 2 subfamily, such as D3 and D4, or of the Di subfamily, such as O and D5 with specificity. for D 2 only and not for D ^ and D 2 .
Les souris D2 - / - développent des hypertrophies des lobes intermédiaires et antérieurs de l'hypophyse, ces phénomènes étant accompagnés d'une synthèse accrue des hormones issues de la transcription du gène de la propiomelanocortine, et de la prolactine. Ces souris D2 - / -, et surtout les femelles, développent aussi des tumeurs ressemblant aux prolactinomes dans une phase plus tardive de leur vie (après 8 mois de la naissance). L'analyse très détaillée des ces phénomènes permet de savoir quelles sont les causes de cette croissance désordonnée des cellules hypophysaires et de trouver des traitements capables de les prévenir.D 2 - / - mice develop hypertrophies of the intermediate and anterior lobes of the pituitary gland, these phenomena being accompanied by an increased synthesis of the hormones resulting from the transcription of the propiomelanocortin gene, and of prolactin. These D 2 - / - mice, and especially the females, also develop prolactinoma-like tumors in a later phase of their life (after 8 months of birth). A very detailed analysis of these phenomena allows us to know what are the causes of this disorderly growth of pituitary cells and to find treatments capable of preventing them.
Le récepteur dopaminergique étant très répandu au niveau central, hypophysaire, mais aussi au niveau du système olfactif, dans la rétine, etc.. l'invention selon un mode de réalisation avantageux, concerne un modèle, pour l'étude de pathologies humaines dues à un manque ou à une réduction de récepteur D .The dopaminergic receptor being very widespread at the central, pituitary, but also at the level of the olfactory system, in the retina, etc. the invention according to an advantageous embodiment, relates to a model, for the study of human pathologies due to a lack or reduction of the D receptor.
En ce qui concerne les souris qui n'ont plus la forme longue du récepteur D2, les D2L - / - , elles ont clairement un phénotype différent des D - / -. Le phénotype consiste en une hyperlocomotion par rapport aux souris sauvages. Ces souris vont permettre d'assigner la fonction de chaque isoforme du récepteur D2 in vivo. La forme D2S pouvant représenter l' auto-récepteur dopaminergique, c'est à dire le récepteur qui contrôle la synthèse de la dopamine, l'intérêt industriel des animaux de l'invention consiste en leur utilisation pour tester des composés n'agissant que sur la forme D2S du récepteur D2 et permettant de régler la synthèse de la dopamine. L'invention concerne également des animaux qui sont générés par croisement entre les souris D2 - / - et D L - / - avec d'autres animaux mutants, vis-à-vis d'autres gènes de récepteurs ou de molécules intervenant dans la voie métabolique dopaminergique.As for the mice which no longer have the long form of the D 2 receptor, the D 2 L - / -, they clearly have a phenotype different from the D - / -. The phenotype consists of hyperlocomotion compared to wild mice. These mice will make it possible to assign the function of each isoform of the D 2 receptor in vivo. As the D 2 S form can represent the dopaminergic auto-receptor, that is to say the receptor which controls the synthesis of dopamine, the industrial interest of the animals of the invention consists in their use for testing compounds which do not act on the D 2 O and D 2 receptor for adjusting the synthesis of dopamine. The invention also relates to animals which are generated by crossing D 2 - / - and DL - / - mice with other mutant animals, vis-à-vis other receptor genes or molecules involved in the pathway. dopaminergic metabolic.
L'invention concerne également l'utilisation des souris de l'invention pour tester des vecteurs utilisés pour la thérapie génique de maladies telles que la maladie de Parkinson, la schizophrénie et d'autres maladies neurodégénératives .
The invention also relates to the use of the mice of the invention for testing vectors used for gene therapy of diseases such as Parkinson's disease, schizophrenia and other neurodegenerative diseases.
Claims
1. Utilisation d'un animal mammifère transgénique non humain dont l'expression du gène codant pour le récepteur dopaminergique D2 est modifiée, notamment sur les cellules lactotropes de l'hypophyse, par rapport à une expression normale, notamment sur les cellules lactotropes de l'hypophyse, pour la détermination d'un médicament actif sur les pathologies impliquant le récepteur D2.1. Use of a non-human transgenic mammalian animal whose expression of the gene coding for the dopaminergic receptor D 2 is modified, in particular on the lactotropic cells of the pituitary gland, compared to a normal expression, in particular on the lactotropic cells of pituitary gland, for the determination of a drug active on pathologies involving the D 2 receptor.
2. Utilisation selon la revendication 1, d'un animal mammifère transgénique non humain susceptible de surexprimer, notamment sur les cellules lactotropes de l'hypophyse, l'isoforme D2L et/ou l'isoforme D2S du récepteur dopaminergique D2.2. Use according to claim 1, of a non-human transgenic mammal animal capable of overexpressing, in particular on the lactotropic cells of the pituitary gland, the isoform D 2 L and / or the isoform D 2 S of the dopaminergic receptor D 2 .
3. Utilisation selon la revendication 1, d'un animal mammifère transgénique non humain qui n'exprime plus l'isoforme D2L, ou qui n'exprime plus ni l'isoforme D2L, ni l'isoforme D2S.3. Use according to claim 1, of a non-human transgenic mammalian animal which no longer expresses the isoform D 2 L, or which no longer expresses either the isoform D 2 L or the isoform D 2 S.
4. Animal mammifère transgénique non humain ou cellules de mammifères contenant dans leur génome une construction choisie parmi:4. Non-human transgenic mammal animal or mammalian cells containing in their genome a construct chosen from:
- un promoteur permettant la surexpression de l'isoforme D2L sur les cellules lactotropes de l'hypophyse, notamment le promoteur de la prolactine, + ADNc de D2L + un terminateur de transcription, notamment une partie du génome du virus SV40 contenant le site de polyadénylation, - un promoteur permettant la surexpression de l'isoforme D2S sur les cellules lactotropes de l'hypophyse, notamment le promoteur de la prolactine, + ADNc de D2S + un terminateur de transcription, notamment une partie du génome du virus SV40 contenant le site de polyadénylation,- a promoter allowing the overexpression of the D 2 L isoform on the lactotropic cells of the pituitary gland, in particular the prolactin promoter, + D 2 L cDNA + a transcription terminator, in particular a part of the genome of the SV40 virus containing the polyadenylation site, a promoter allowing the overexpression of the D 2 S isoform on the lactotropic cells of the pituitary gland, in particular the prolactin promoter, + D 2 S cDNA + a transcription terminator, in particular part of the SV40 virus genome containing the polyadenylation site,
- un promoteur permettant la surexpression de l'isoforme D2L sur les cellules lactotropes de l'hypophyse, notamment le promoteur de la prolactine,a promoter allowing the overexpression of the D 2 L isoform on the lactotropic cells of the pituitary gland, in particular the prolactin promoter,
+ ADNc de D2L + un terminateur de transcription, notamment une partie du génome du virus SV40 contenant le site de polyadénylation, et un promoteur permettant la surexpression de l'isoforme D2S sur les cellules lactotropes de l'hypophyse, notamment le promoteur de la prolactine, + ADNc de D2S + un terminateur de transcription, notamment une partie du génome du virus+ D 2 L cDNA + a transcription terminator, in particular a part of the SV40 virus genome containing the polyadenylation site, and a promoter allowing the overexpression of the D 2 S isoform on the lactotropic cells of the pituitary gland, in particular the prolactin promoter, + D 2 S cDNA + a transcription terminator, in particular a part of the virus genome
SV40 contenant le site de polyadénylation. SV40 containing the polyadenylation site.
5. Animal mammifère transgénique non humain contenant l'une des constructions selon la revendication 4, et dont les cellules lactotropes surexpriment D2L et/ou de D2S.5. A non-human transgenic mammal animal containing one of the constructs according to claim 4, and whose lactotropic cells overexpress D 2 L and / or D 2 S.
6. Animal mammifère transgénique non humain ou cellules de mammifères contenant le gène du récepteur D2 dans lequel un fragment du gène de D2 contenant l'exon 2 ou un fragment du gène de D2 contenant l'exon 6 est remplacé par tout ou partie d'un gène marqueur sous le contrôle d'un promoteur approprié, notamment le gène de résistance à la néomycine (neo) sous le contrôle du promoteur phosphoglycérate kinase -1 (PGK-1).6. Non-human transgenic mammal animal or mammalian cells containing the D 2 receptor gene in which a fragment of the D 2 gene containing exon 2 or a fragment of the D 2 gene containing exon 6 is replaced by all or part of a marker gene under the control of an appropriate promoter, in particular the neomycin resistance gene (neo) under the control of the phosphoglycerate kinase -1 promoter (PGK-1).
7. Animal mammifère transgénique non humain contenant l'une des constructions selon la revendication 6, et dans lequel soit l'expression de D2L est supprimée, l'expression normale de D S étant maintenue, soit l'expression de D2L et de D2S est supprimée.7. A non-human transgenic mammal animal containing one of the constructs according to claim 6, and in which either the expression of D 2 L is suppressed, the normal expression of DS being maintained, or the expression of D 2 L and of D 2 S is deleted.
8. Cellules cultivées à partir des animaux mammifères transgéniques non humains selon l'une des revendications 4 ou 6.8. Cells cultured from non-human transgenic mammalian animals according to one of claims 4 or 6.
9. Mammifère transgénique non humain produit par introduction, notamment microinjection dans un ovocyte fécondé, de l'une des constructions selon la revendication 4, insertion de l'embryon dans une mère de substitution et développement de l'embryon à terme.9. A non-human transgenic mammal produced by the introduction, in particular microinjection into a fertilized oocyte, of one of the constructions according to claim 4, insertion of the embryo into a surrogate mother and development of the term embryo.
10. Mammifère transgénique non humain obtenu par introduction dans un blastocyte de cellules souches embryonnaires (cellules ES) comportant dans leur génome l'une des constructions selon la revendication 6, obtenu par recombinaison homologue, sélection d'animaux chimères mâles selon un critère correspondant à la lignée ES, croisement des animaux sélectionnés avec des souris, notamment des souris C57 Black 6, pour obtenir des animaux hétérozygotes par rapport à l'une des constructions selon la revendication 6 et éventuellement croisement de deux hétérozygotes pour obtenir un animal homozygote par rapport à l'une des constructions selon la revendication 6.10. Non-human transgenic mammal obtained by introduction into a blastocyte of embryonic stem cells (ES cells) comprising in their genome one of the constructions according to claim 6, obtained by homologous recombination, selection of male chimeric animals according to a criterion corresponding to the ES line, crossing of selected animals with mice, in particular C57 Black 6 mice, to obtain animals heterozygous with respect to one of the constructions according to claim 6 and optionally crossing of two heterozygotes to obtain an animal homozygous with respect to one of the constructions according to claim 6.
11. Mammifère selon l'une des revendications 4 ou 6, produit par croisement d'animaux transgéniques exprimant différentes constructions. 11. Mammal according to one of claims 4 or 6, produced by crossing transgenic animals expressing different constructions.
12. Procédé d'obtention d'un modèle transgénique pour l'étude des pathologies impliquant les récepteurs D2, et de leur traitement, comprenant l'introduction dans les cellules ou des embryons de mammifères non humains d'une construction choisie parmi: - un promoteur permettant la surexpression de l'isoforme D2L sur les cellules lactotropes des glandes pituitaires, notamment le promoteur de la prolactine, + ADNc de D2L + un terminateur de transcription, notamment une partie du génome du virus SV40 contenant le site de polyadénylation et en particulier fragment BamHI-HindII(-3000/+33) du promoteur de la prolactine + fragment Smal-EcoRV de l'ADNc de D2L + fragment BamHI-BglII de12. Method for obtaining a transgenic model for the study of pathologies involving D 2 receptors, and their treatment, comprising the introduction into the cells or embryos of non-human mammals of a construction chosen from: - a promoter allowing the overexpression of the D 2 L isoform on the lactotropic cells of the pituitary glands, in particular the prolactin promoter, + D 2 L cDNA + a transcription terminator, in particular a part of the genome of the SV40 virus containing the site of polyadenylation and in particular BamHI-HindII fragment (-3000 / + 33) of the prolactin promoter + Smal-EcoRV fragment of D 2 L cDNA + BamHI-BglII fragment of
SV40,SV40,
- un promoteur permettant la surexpression de l'isoforme D2S sur les cellules lactotropes des glandes pituitaires, notamment le promoteur de la prolactine, -I- ADNc de D2S + un terminateur de transcription, notamment une partie du génome du virus SV40 contenant le site de polyadénylation et en particulier fragment BamHI-HindII(-3000/+33) du promoteur de la prolactine + fragment Smal-EcoRV de l'ADNc de D2S + fragment BamHI-BglII de SV40,- a promoter allowing the overexpression of the isoform D 2 S on the lactotropic cells of the pituitary glands, in particular the promoter of prolactin, -I- cDNA of D 2 S + a transcription terminator, in particular a part of the genome of the SV40 virus containing the polyadenylation site and in particular the BamHI-HindII fragment (-3000 / + 33) of the prolactin promoter + Smal-EcoRV fragment of the D 2 S cDNA + BamHI-BglII fragment of SV40,
- un promoteur permettant la surexpression de l'isoforme D2L sur les cellules lactotropes des glandes pituitaires, notamment le promoteur de la prolactine, + ADNc de D2L + un terminateur de transcription, notamment une partie du génome du virus SV40 contenant le site de polyadénylation et en particulier fragment BamHI-HindII(-3000/+33) du promoteur de la prolactine, + fragment Smal-EcoRV de l'ADNc de D2L + fragment BamHI-Bgiπ de SV40, et un promoteur permettant la surexpression de l'isoforme D2S sur les cellules lactotropes des glandes pituitaires, notamment le promoteur de la prolactine, + ADNc de D2S + un terminateur de transcription, notamment une partie du génome du virus SV40 contenant le site de polyadénylation et en particulier fragment BamHI-HindII(-3000/+33) du promoteur de la prolactine + fragment Smal-EcoRV de l'ADNc de D2S + fragment BamHI-BglII dea promoter allowing the overexpression of the D 2 L isoform on the lactotropic cells of the pituitary glands, in particular the prolactin promoter, + D 2 L cDNA + a transcription terminator, in particular a part of the genome of the SV40 virus containing the polyadenylation site and in particular BamHI-HindII fragment (-3000 / + 33) of the prolactin promoter, + Smal-EcoRV fragment of D 2 L cDNA + BamHI-Bgiπ fragment of SV40, and a promoter allowing overexpression of the D 2 S isoform on the lactotropic cells of the pituitary glands, in particular the prolactin promoter, + D 2 S cDNA + a transcription terminator, in particular a part of the SV40 virus genome containing the polyadenylation site and in particular BamHI-HindII fragment (-3000 / + 33) of the prolactin promoter + Smal-EcoRV fragment of D 2 S cDNA + BamHI-BglII fragment of
SV40,SV40,
- le gène de D2 dans lequel un fragment contenant l'exon 2 est remplacé par un gène marqueur sous le contrôle d'un promoteur approprié, notamment une cassette contenant le gène neo sous le contrôle du promoteur PGKI, et en particulier le gène de D2 dans lequel le fragment génomique de 0,9 Kb Ncol, contenant l'exon 2 est remplacé par la cassette PGK-neo,the gene for D 2 in which a fragment containing exon 2 is replaced by a marker gene under the control of an appropriate promoter, in particular a cassette containing the neo gene under the control of the PGKI promoter, and in particular the gene for D 2 in which the 0.9 Kb Ncol genomic fragment containing exon 2 is replaced by the PGK-neo cassette,
- le gène de D2 dans lequel un fragment contenant l'exon 6 est remplacé par un gène marqueur sous le contrôle d'un promoteur approprié, notamment par une cassette contenant le gène neo sous le contrôle du promoteur PGKI, et en particulier le gène de D2 dans lequel le fragment génomique BglII-BglII contenant l'exon 6 est remplacé par la cassette PGK-neo.- the D 2 gene in which a fragment containing exon 6 is replaced by a marker gene under the control of an appropriate promoter, in particular by a cassette containing the neo gene under the control of the PGKI promoter, and in particular the D 2 gene in which the BglII-BglII genomic fragment containing exon 6 is replaced by the PGK-neo cassette.
13. Procédé de criblage de médicaments actifs sur des pathologies impliquant les récepteurs D2, notamment les prolactinomes ou certaines pathologies neurodégénératives telles que la maladie de Parkinson, la maladie d'Huntington ou la schizophrénie comprenant:13. A method of screening for drugs active on pathologies involving D 2 receptors, in particular prolactinomas or certain neurodegenerative pathologies such as Parkinson's disease, Huntington's disease or schizophrenia comprising:
* l'administration à un mammifère non humain transgénique ou à des cellules de mammifères non humains transgéniques contenant une construction choisie parmi:* administration to a transgenic non-human mammal or to cells of transgenic non-human mammals containing a construct chosen from:
- un promoteur permettant la surexpression de l'isoforme D2L sur les cellules lactotropes des glandes pituitaires, notamment le promoteur de la prolactine, + ADNc de D2L + un terminateur de transcription, notamment une partie du génome du virus SV40 contenant le site de polyadénylation et en particulier fragment BamHI-HindII(-3O00/+33) du promoteur de la prolactine + fragment Smal-EcoRV de l'ADNc de D2L -I- fragment BamHI-BglII de SV40,a promoter allowing the overexpression of the D 2 L isoform on the lactotropic cells of the pituitary glands, in particular the prolactin promoter, + D 2 L cDNA + a transcription terminator, in particular a part of the genome of the SV40 virus containing the polyadenylation site and in particular BamHI-HindII fragment (-3O00 / + 33) of the prolactin promoter + Smal-EcoRV fragment of D 2 L cDNA -I- BamHI-BglII fragment of SV40,
- un promoteur permettant la surexpression de l'isoforme D2S sur les cellules lactotropes des glandes pituitaires, notamment le promoteur de la prolactine, + ADNc de D2S + un terminateur de transcription, notamment une partie du génome du virus SV40 contenant le site de polyadénylation et en particulier fragment BamHI-HindII(-30O0/+33) du promoteur de la prolactine + fragment Smal-EcoRV de l'ADNc de D2S + fragment BamHI-BglII de SV40,a promoter allowing the overexpression of the D 2 S isoform on the lactotropic cells of the pituitary glands, in particular the prolactin promoter, + D 2 S cDNA + a transcription terminator, in particular a part of the genome of the SV40 virus containing the polyadenylation site and in particular BamHI-HindII fragment (-30O0 / + 33) of the prolactin promoter + Smal-EcoRV fragment of D 2 S cDNA + BamHI-BglII fragment of SV40,
- un promoteur permettant la surexpression de l'isoforme D2L sur les cellules lactotropes des glandes pituitaires, notamment le promoteur de la prolactine, + ADNc de D2L + un terminateur de transcription, notamment une partie du génome du virus SV40 contenant le site de polyadénylation et en particulier fragment BamHI-HindII(-30O0/+33) du promoteur de la prolactinea promoter allowing the overexpression of the D 2 L isoform on the lactotropic cells of the pituitary glands, in particular the prolactin promoter, + D 2 L cDNA + a transcription terminator, in particular a part of the genome of the SV40 virus containing the polyadenylation site and in particular BamHI-HindII fragment (-30O0 / + 33) of the prolactin promoter
+ fragment Smal-EcoRV de l'ADNc de D2L + fragment BamHI-BglII de SV40, et un promoteur permettant la surexpression de l'isoforme D2S sur les cellules lactotropes des glandes pituitaires, notamment le promoteur de la prolactine, + ADNc de D2S + un terminateur de transcription, notamment une partie du génome du virus SV40 contenant le site de polyadénylation et en particulier fragment BamHI-HindII(-3OO0/+33) du promoteur de la prolactine + fragment Smal-EcoRV de l'ADNc de D2S + fragment BamHI-BglII de SV40, - le gène de D2 dans lequel un fragment contenant l'exon 2 est remplacé par un gène marqueur sous le contrôle d'un promoteur approprié, notamment une cassette contenant le gène neo sous le contrôle du promoteur PGKI, et en particulier le gène de D2 dans lequel le fragment génomique de 0,9 Kb Ncol, contenant l'exon 2 est remplacé par la cassette PGK-neo,+ Smal-EcoRV fragment of the D 2 L cDNA + BamHI-BglII fragment of SV40, and a promoter allowing the overexpression of the D 2 S isoform on the lactotropic cells of the pituitary glands, in particular the prolactin promoter, + CDNA of D 2 S + a transcription terminator, in particular a part of the genome of the SV40 virus containing the polyadenylation site and in particular BamHI-HindII fragment (-3OO0 / + 33) of the prolactin promoter + Smal-EcoRV fragment of the CDNA of D 2 S + fragment BamHI-BglII of SV40, the gene for D 2 in which a fragment containing exon 2 is replaced by a marker gene under the control of an appropriate promoter, in particular a cassette containing the neo gene under the control of the PGKI promoter, and in particular the gene for D 2 in which the 0.9 Kb Ncol genomic fragment containing exon 2 is replaced by the PGK-neo cassette,
- le gène de D2 dans lequel un fragment contenant l'exon 6 est remplacé par un gène marqueur sous le contrôle d'un promoteur approprié, notamment par une cassette contenant le gène neo sous le contrôle du promoteur PGKI, et en particulier le gène de D2 dans lequel le fragment génomique BglII-BglII contenant l'exon 6 est remplacé par la cassette PGK-neo du composé à tester,- the D 2 gene in which a fragment containing exon 6 is replaced by a marker gene under the control of an appropriate promoter, in particular by a cassette containing the neo gene under the control of the PGKI promoter, and in particular the gene D 2 in which the BglII-BglII genomic fragment containing exon 6 is replaced by the PGK-neo cassette of the compound to be tested,
* et la détermination de la diminution du taux de prolactine (dans le cas des prolactinomes) ou de l'amélioration de l'état neurodégénératif (dans le cas des pathologies neurodégénératives).* and the determination of the decrease in the level of prolactin (in the case of prolactinomas) or the improvement of the neurodegenerative state (in the case of neurodegenerative pathologies).
14. Procédé de criblage de médicaments actifs sur un modèle animal où les taux de prolactine sont diminués à l'aide d'animaux transgéniques surexprimant D S, comprenant l'administration à un mammifère non humain transgénique ou à des cellules de mammifères non humains transgéniques contenant une construction choisie parmi:14. A method of screening for drugs active in an animal model in which prolactin levels are reduced using transgenic animals overexpressing DS, comprising administering to a transgenic non-human mammal or cells of transgenic non-human mammals containing a construction chosen from:
- un promoteur permettant la surexpression de l'isoforme D2S sur les cellules lactotropes des glandes pituitaires, notamment le promoteur de la prolactine, + ADNc de D2S + un terminateur de transcription, notamment une partie du génome du virus SV40 contenant le site de polyadénylation et en particulier fragment BamHI-HindII(-30O0/+33) du promoteur de la prolactine + fragment Smal-EcoRV de l'ADNc de D2S + fragment BamHI- BglII de SV40, du composé à tester,a promoter allowing the overexpression of the D 2 S isoform on the lactotropic cells of the pituitary glands, in particular the prolactin promoter, + D 2 S cDNA + a transcription terminator, in particular a part of the genome of the SV40 virus containing the polyadenylation site and in particular BamHI-HindII fragment (-30O0 / + 33) of the prolactin promoter + SmaI-EcoRV fragment of D 2 S cDNA + BamHI-BglII fragment of SV40, of the compound to be tested,
* et la détermination de l'augmentation du taux de prolactine.* and the determination of the increase in prolactin level.
15. Procédé de criblage de médicaments actifs sur des pathologies neurodégénératives telles que la maladie de Parkinson, la maladie d'Huntington ou la schizophrénie à l'aide d'animaux transgéniques dans lesquels D2S et D2L ne sont plus exprimés, comprenant:15. Method for screening for drugs active on neurodegenerative pathologies such as Parkinson's disease, Huntington's disease or schizophrenia using transgenic animals in which D 2 S and D 2 L are no longer expressed, comprising :
* l'administration à un mammifère non humain transgénique ou à des cellules de mammifères non humains transgéniques contenant une construction choisie parmi: - le gène de D2 dans lequel un fragment contenant l'exon 2 est remplacé par un gène marqueur sous le contrôle d'un promoteur approprié, notamment une cassette contenant le gène neo sous le contrôle du promoteur PGKI, et en particulier le gène de D2 dans lequel le fragment génomique de 0,9 Kb Ncol, contenant l'exon 2 est remplacé par la cassette PGK-neo du composé à tester,* administration to a transgenic non-human mammal or to cells of transgenic non-human mammals containing a construct chosen from: the gene for D 2 in which a fragment containing exon 2 is replaced by a marker gene under the control of an appropriate promoter, in particular a cassette containing the neo gene under the control of the PGKI promoter, and in particular the gene for D 2 in which the 0.9 Kb Ncol genomic fragment containing exon 2 is replaced by the PGK-neo cassette of the compound to be tested,
* et la détermination de l'amélioration de l'état neurodégénératif.* and the determination of the improvement of the neurodegenerative state.
16. Construction transgénique choisie parmi: - un promoteur permettant la surexpression de l'isoforme D2L sur les cellules lactotropes de l'hypophyse, notamment le promoteur de la prolactine, -I- ADNc de D2L + un terminateur de transcription, notamment une partie du génome du virus SV40 contenant le site de polyadénylation et en particulier fragment BamHI-HindII(-3000/-t-33) du promoteur de la prolactine + fragment Smal-EcoRV de l'ADNc de D2L + fragment BamHI-BglII de SV40,16. Transgenic construct chosen from: - a promoter allowing the overexpression of the D 2 L isoform on the lactotropic cells of the pituitary gland, in particular the prolactin promoter, -I- D 2 L cDNA + a transcription terminator, in particular a part of the SV40 virus genome containing the polyadenylation site and in particular the BamHI-HindII fragment (-3000 / -t-33) of the prolactin promoter + Smal-EcoRV fragment of the D 2 L cDNA + BamHI fragment -BglII of SV40,
- un promoteur permettant la surexpression de l'isoforme D2S sur les cellules lactotropes de l'hypophyse, notamment le promoteur de la prolactine, + ADNc de D2S + un terminateur de transcription, notamment une partie du génome du virus SV40 contenant le site de polyadénylation et en particulier fragment BamHI-HindII(-3000/+33) du promoteur de la prolactine + fragment Smal-EcoRV de l'ADNc de D2S + fragment BamHI-BglII de SV40,- a promoter allowing the overexpression of the isoform D 2 S on the lactotropic cells of the pituitary gland, in particular the promoter of prolactin, + cDNA of D 2 S + a transcription terminator, in particular a part of the genome of the SV40 virus containing the polyadenylation site and in particular the BamHI-HindII fragment (-3000 / + 33) of the prolactin promoter + Smal-EcoRV fragment of the D 2 S cDNA + BamHI-BglII fragment of SV40,
- un promoteur permettant la surexpression de l'isoforme D2L sur les cellules lactotropes de l'hypophyse, notamment le promoteur de la prolactine, + ADNc de D2L + un terminateur de transcription, notamment une partie du génome du virus SV40 contenant le site de polyadénylation et en particulier fragment BamHI-HindII(-3000/-t-33) du promoteur de la prolactine + fragment Smal-EcoRV de l'ADNc de D2L + fragment BamHI-BglII de SV40, et un promoteur permettant la surexpression de l'isoforme D2S sur les cellules lactotropes de l'hypophyse, notamment le promoteur de la prolactine, + ADNc de D2S + un terminateur de transcription, notamment une partie du génome du virus SV40 contenant le site de polyadénylation et en particulier fragment BamHI-HindII(-30OO/-l-33) du promoteur de la prolactine + fragment Smal-EcoRV de l'ADNc de D2S + fragment BamHI-BglII de SV40,- a promoter allowing the overexpression of the D 2 L isoform on the lactotropic cells of the pituitary gland, in particular the prolactin promoter, + D 2 L cDNA + a transcription terminator, in particular a part of the genome of the SV40 virus containing the polyadenylation site and in particular BamHI-HindII fragment (-3000 / -t-33) of the prolactin promoter + Smal-EcoRV fragment of D 2 L cDNA + BamHI-BglII fragment of SV40, and a promoter allowing overexpression of the D 2 S isoform on the lactotropic cells of the pituitary gland, in particular the prolactin promoter, + D 2 S cDNA + a transcription terminator, in particular a part of the SV40 virus genome containing the polyadenylation site and in particular BamHI-HindII fragment (-30OO / -l-33) of the prolactin promoter + Smal-EcoRV fragment of the D 2 S cDNA + BamHI-BglII fragment of SV40,
- le gène de D2 dans lequel un fragment contenant l'exon 2 est remplacé par un gène marqueur sous le contrôle d'un promoteur approprié, notamment une cassette contenant le gène neo sous le contrôle du promoteur PGKI, et en particulier le gène de D2 dans lequel le fragment génomique de 0,9 Kb Ncol, contenant l'exon 2 est remplacé par la cassette PGK-neo, - le gène de D2 dans lequel un fragment contenant l'exon 6 est remplacé par un gène marqueur sous le contrôle d'un promoteur approprié, notamment par une cassette contenant le gène neo sous le contrôle du promoteur PGKI, et en particulier le gène de D2 dans lequel le fragment génomique Bglϋ-BglII contenant l'exon 6 est remplacé par la cassette PGK-neo. the gene for D 2 in which a fragment containing exon 2 is replaced by a marker gene under the control of an appropriate promoter, in particular a cassette containing the neo gene under the control of the PGKI promoter, and in particular the gene for D 2 in which the 0.9 Kb Ncol genomic fragment containing exon 2 is replaced by the PGK-neo cassette, - the D 2 gene in which a fragment containing exon 6 is replaced by a marker gene under the control of an appropriate promoter, in particular by a cassette containing the neo gene under the control of the PGKI promoter, and in particular the gene of D 2 in which the Bglϋ-BglII genomic fragment containing exon 6 is replaced by the PGK-neo cassette.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR95/05013 | 1995-04-26 | ||
| FR9505013A FR2733514A1 (en) | 1995-04-26 | 1995-04-26 | NON-HUMAN TRANSGENIC ANIMAL IN WHICH D2 RECEPTOR EXPRESSION IS MODIFIED |
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| Publication Number | Publication Date |
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| WO1996034094A1 true WO1996034094A1 (en) | 1996-10-31 |
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| PCT/FR1996/000624 WO1996034094A1 (en) | 1995-04-26 | 1996-04-24 | Non-human transgenic animal with modified d2 receptor expression |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998035989A1 (en) * | 1997-02-14 | 1998-08-20 | Toernell Jan | Method for screening and transgenic model |
| WO2004082570A2 (en) * | 2003-03-17 | 2004-09-30 | Bayer Healthcare Ag | Diagnostics and therapeutics for diseases associated with dopamine receptor d2 (drd2) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1989001039A1 (en) * | 1987-08-06 | 1989-02-09 | Edison Animal Biotechnology Center | A method of directing transgenic expression in animals using a prolactin promoter |
| WO1990005780A1 (en) * | 1988-11-18 | 1990-05-31 | State Of Oregon, Acting By And Through The Oregon State Board Of Higher Education, Acting For And On Behalf Of The Oregon Health Sciences University | Dopamine receptors and genes |
-
1995
- 1995-04-26 FR FR9505013A patent/FR2733514A1/en not_active Withdrawn
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1996
- 1996-04-24 WO PCT/FR1996/000624 patent/WO1996034094A1/en active Application Filing
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1989001039A1 (en) * | 1987-08-06 | 1989-02-09 | Edison Animal Biotechnology Center | A method of directing transgenic expression in animals using a prolactin promoter |
| WO1990005780A1 (en) * | 1988-11-18 | 1990-05-31 | State Of Oregon, Acting By And Through The Oregon State Board Of Higher Education, Acting For And On Behalf Of The Oregon Health Sciences University | Dopamine receptors and genes |
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| "Symposium 2 : Use of transgenic animals im pharmacology", JOINT MEETING OF THE DEUTSCHE GESELLSCHAFT FÜR PHARMAKOLOGIE UND TOXIKOLOGIE AND THE ASSOCIATION FRANAISE DES PHARMACOLOGISTES, 6 October 1993 (1993-10-06) - 8 October 1993 (1993-10-08), UNIVERSITÉ DE LILLE, FRANCE * |
| BAIK, J-H., ET AL.: "Parkinsonian-like locomotor impairment in mice lacking dopamine D2 receptors", NATURE, vol. 377, no. 6548, 5 October 1995 (1995-10-05), LONDON GB, pages 424 - 428, XP002011018 * |
| FITZGERALD, L.W. ET AL.: "Asymmetric elevation of striatal dopamine D2 receptors in the chakragati mouse: neurobehavioral dysfunction in a transgenic insertional mutant", BRAIN RESEARCH, vol. 580, no. 1-2, 15 May 1992 (1992-05-15), pages 18 - 26, XP000601511 * |
| GUIRAMAND, J. ET AL.: "Alternative splicing of the Dopamine D2 Receptor directs specificity of coupling to G-proteins", JOURNAL OF BIOLOGICAL CHEMISTRY (MICROFILMS), vol. 270, no. 13, 31 March 1995 (1995-03-31), MD US, pages 7354 - 7358, XP002011021 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998035989A1 (en) * | 1997-02-14 | 1998-08-20 | Toernell Jan | Method for screening and transgenic model |
| WO2004082570A2 (en) * | 2003-03-17 | 2004-09-30 | Bayer Healthcare Ag | Diagnostics and therapeutics for diseases associated with dopamine receptor d2 (drd2) |
| WO2004082570A3 (en) * | 2003-03-17 | 2005-01-27 | Bayer Healthcare Ag | Diagnostics and therapeutics for diseases associated with dopamine receptor d2 (drd2) |
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| FR2733514A1 (en) | 1996-10-31 |
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