WO2007083367A1 - Nonhuman mammal capable of expressing reporter gene specifically to inhibitory neuron - Google Patents

Abstract

There is provided an animal capable of efficient observation and analysis of inhibitory neuron by realizing retention, on a chromosome of nonhuman mammal, of a reporter gene expressibly linked to a DNA fragment containing a 5’ expression regulating region of about 100 kbp of vesicular GABA transporter gene.

Description

 Specification

 Non-human mammals that express reporter genes specifically for inhibitory neurons

 [0001] The present invention relates to a non-human mammal that expresses a reporter gene such as a fluorescent protein gene in a suppressive-euron-specific manner.

 Background art

 [0002] The brain is made up of a collection of neural networks composed of excitability-euron and inhibitory-euron! Inhibitory-Euron's main neurotransmitters (inhibitory neurotransmitters) are γ-aminobutyric acid (GABA) and glycine. Release GABA as a neurotransmitter Nerve cells are GABA neurons, which are widely distributed in the brain and spinal cord. On the other hand, neurons that release dalysin are glycine-euron and are distributed mainly in the brain stem and spinal cord. Inhibitor transmitters are known to control neuronal potential activity and play a central role in building brain functions such as arousal, sleep, circadian rhythm and learning, movement, and sensory information processing. ! / GABA has been reported to be associated with neuropsychiatric disorders such as epilepsy and alcohol psychosis, and mental symptoms such as anxiety and depression. On the other hand, glycine is known to be associated with startle disease.

 Therefore, (l) clarifying the mechanism of action of GABA and glycine, and (2) clarifying the function of inhibitory-euron (GABA-euron and glycine-euron) with GABA and glycine as transmitters. Understand the function of the brain and nerves, contribute to the elucidation of the pathogenesis of the above diseases and the establishment of treatments.

 GABA and glycine are accumulated in synaptic vesicles by the vesicular GABA transporter (VGAT) and then released into the synaptic cleft. Since VGAT is specifically expressed in inhibitory-euron (GABA and europium and glycine-euron), VGAT is also used as one of the most effective inhibitory-euron. On the other hand, inhibitory-eurone is generally scattered in the central nervous system and is relatively small in number, so it was difficult to accurately identify it in the living body and to analyze its functions such as electrophysiological characteristics.

[0003] Inhibitory-Green fluorescent protein gene is a glutamic acid that visualizes euron. A transgenic animal having been inserted into the same reading frame as the exon of the minate decarboxylase gene has been reported (Patent Document 1).

 An animal having a gene encoding a fluorescent protein linked so as to be under the control of the expression control region of the VGAT gene has also been reported (Patent Document 2). In Patent Document 2, a region of about 8 kbp of the 5 ′ expression regulatory region was used as the expression control region of the VGAT gene. However, it has been desired to develop an animal that can visualize inhibitory neurons more efficiently than the expression level of fluorescent protein in the animals described in these documents.

 Patent Document 1: Japanese Patent Laid-Open No. 2003-088272

 Patent Document 2: Japanese Patent Laid-Open No. 2003-204796

 Disclosure of the invention

 [0004] An object of the present invention is to provide an animal capable of efficiently observing and analyzing inhibitory-euron.

 [0005] The present inventors diligently studied to solve the above problems. As a result, by using a non-human mammal in which a reporter gene operably linked to a DNA fragment containing a 5 'expression regulatory region of about 10 LOOkbp of the VGAT gene is retained on the chromosome, it is The inventors have found that Euron can be observed and analyzed efficiently, and have completed the present invention.

 That is, the present invention is as follows.

 (1) A non-human mammal that retains on a chromosome a reporter gene operably linked to a DNA fragment containing a 5 'expression regulatory region of about 10 OOkbp of the vesicular GABA transporter gene.

 (2) The non-human mammal according to (1), wherein the reporter gene is a gene in which the 3 ′ expression regulatory region of the vesicular GABA transporter gene is further linked to the 3 ′ side thereof.

 (3) a gene containing a vesicular GABA transporter gene containing a 5 'expression regulatory region of about lOOkbp and a reporter gene inserted at the position of the translation initiation codon of the gene. ) Non-human mammals.

(4) The non-human mammal of any one of (1) to (3), wherein the reporter gene is a Venus gene Milk animals.

 (5) The non-human mammal according to any one of (1) to (4), which is a rat.

 (6) Addition of a test substance to the non-human mammal of any one of (1) to (5) or to a nerve tissue that can also obtain the animal's power, and suppressive-suppressive using the expression level of the reporter gene in euron as an indicator -A screening method for a neurotransmission-regulating agent, comprising selecting a substance having an activity of controlling neurotransmission from euron.

 Brief Description of Drawings

 [0007] FIG. 1 is an explanatory diagram of a BAC clone containing a VGAT gene (wild-type BAC), a targeting construct for homologous recombination, and a BAC clone modified by homologous recombination (modified BAC).

 FIG. 2 is a diagram (photograph) showing the results of observation of the brain of a VGAT-venus rat with a fluorescence microscope.

 [Fig. 3] Diagram showing the results of immunostaining of VGAT-venus rat cerebral cortex specimens with anti-GFP antibody

(Photo). Upper part is brain membrane and layer I, middle part is II ~ V layer, lower part is VI layer and white matter.

[Fig. 4] Diagram showing the results of VGAT- _venUS rat cerebellar cortex specimens observed with a fluorescence microscope (photograph)

). The upper part is a molecular layer, the central part is a Purkinje cell layer, and the lower part is a granular layer.

 BEST MODE FOR CARRYING OUT THE INVENTION

 [0008] The non-human mammal of the present invention has a reporter gene chromosomally linked to a DNA fragment containing a 5 'expression regulatory region of about 1 OOkbp of the vesicular GABA transporter (VGAT) gene. A non-human mammal that is retained.

 [0009] The VGAT gene is not particularly limited as long as it encodes VGAT, but a mouse-derived gene that prefers a mouse or rat-derived gene is more preferable. The VGAT gene derived from the mouse is registered in the database Ensembl under the registration number ENSMUSG00000037771. The three columns can be found at www.ensembl.org/Mus—musculus/geneview?gene=ENSMUSG00000037771&db=core&—gene—sequence=l.

Examples of the 5 ′ expression regulatory region of about lOOkbp of the VGAT gene include a region of about lOOkbp 5 ′ to the start codon ATG contained in exon 1 of the VGAT gene. In addition, since the base sequence of the mouse V GAT gene is expected to vary depending on the strain and individual, The above region may be one in which one or a plurality of bases are substituted, deleted, inserted, etc., as long as the region induces reporter gene repression—specific expression in euron. Here, the term “multiple” refers to, for example, 2 to 100, preferably 2 to 50, and more preferably 2 to 20. In addition, about lOOkbp is 90-: L 10 kbp, preferably 95-105 kbp, particularly preferably 99-: LOlkbp.

 Moreover, as long as the region of about lOOkbp is included, a region on the 5 ′ side may be further included.

[0010] The non-human mammal of the present invention retains a gene construct that is operably linked to a DNA fragment containing a 5 'expression regulatory region of about lOOkbp of the reporter gene strength VGAT gene. The gene construct may be directly linked to the 5 ′ expression regulatory region, as long as the open reading frame of a single reporter gene is linked downstream of the 5 ′ expression regulatory region of the VGAT gene. , Downstream of the expression regulatory region may be linked through any sequence without inhibiting the expression of the reporter gene. A gene construct in which an open reading frame of a reporter gene, preferably an open reading frame and a polyA sequence is inserted at the position of the translation initiation codon immediately after the 5 ′ expression regulatory region of the VGAT gene is desirable.

 The gene construct contains sequences after the translation initiation codon of the VGAT gene (exons and 3 'expression control regions)! /, Or may! / ヽ. Examples of the 3 ′ expression regulatory region include a region of about 20 kbp downstream of the stop codon of the VGAT gene.

 [0011] DNA containing the 5 'expression regulatory region of the VGAT gene is selected from a genomic library such as BAC, YAC, etc. The clone containing the DNA is selected by a method commonly used, and the 5' expression regulatory region of the VGAT gene Can be obtained by cleaving with a restriction enzyme or the like, or can be obtained by polymerase chain reaction (PCR) or the like. The origin of such DNA is preferably the same type as the host into which it is introduced, but is not limited to this as long as the introduced DNA can function in the host cell! /. An example of a BAC library containing a 5 ′ expression regulatory region of about 10 OOkbp of the VGAT gene is clone # 160L22 available from Genome system (St. Louis, MO, U.S.A.).

[0012] As a reporter gene, a light-emitting or chromogenic protein gene used for selectively visualizing inhibitory-euron, or used for selectively destroying inhibitory-euron. Toxin genes that are present. As a gene encoding a photoprotein, GFP (green fluorescent protein; Prasher, DC, et al., Gene, 111, 229-233 (1992)), a force that changes from green to red when irradiated with ultraviolet rays. (GenBank Accession No. AB085641), a gene encoding a chameleon that emits light depending on the intracellular calcium concentration (GenBank Accession No. AB178711), a Venus gene (Nat Biotechnol. 2 002 Jan; 20 ( l): 87-90.). Examples of the chromogenic protein gene include a gene encoding j8-galactosidase. Examples of the gene encoding the toxin include a gene encoding diphtheria toxin.

 DNA fragments containing these reporter genes can be obtained by amplification by PCR or the like, or commercially available products can be used.

 [0013] The reporter gene can be ligated to the 3 'side of the DNA containing the 5' expression regulatory region of the VGAT gene using a restriction enzyme or the like. A gene construct in which a reporter gene is inserted at the start codon position of the VGAT gene can also be prepared by homologous recombination using a gene construct in which a reporter gene is inserted between sequences before and after the start codon of the VGAT gene. . Since the gene construct has a length of lOOkbp or more, it is preferable to introduce it using BAC (E. coli artificial chromosome).

 [0014] The non-human mammal is more preferably a rat that is preferred for rodents such as mice or rats. (1) There are disease model rats such as GAERS absence seizure rats and Huntington's disease rats. (2) Use of monoclonal antibodies is difficult in mice but possible in rats, and histological analysis is easy. (3) Many physiological experiments have been performed in rats and it is easy to collate the data. (4) Analysis of small brain nuclei in the brainstem is easier in rats than in mice. Particularly preferred.

 [0015] The gene construct prepared as described above is introduced into germ cells of a non-human mammal to produce a genetically modified animal cell, and further this is generated to produce a non-human mammal. it can. Examples of the method for introducing the gene construct into the host include a method of inserting a plurality of copies of the construct into an unspecified position of the genomic DNA of the host, a method of introducing the construct into a specific position of the host genome, and the like.

[0016] As non-human mammal germ cells, fertilized eggs before cleavage are preferably used. like this A fertilized egg is obtained by mating a male and female of a non-human mammal. Although fertilized eggs can be obtained by natural mating, a method of mating with males after artificially adjusting the female sexual cycle of animals is preferred. For example, follicle stimulating hormone (pregnant mare serum gonadotropin; PMSG) and then luteinizing hormone (human ciliary gonadotropin; hCG) For example, by intraperitoneal injection. The dose and interval of administration of these hormones should be determined appropriately according to the type of animal. As described above, fertilized eggs can be obtained by administering hormonal administration to female animals and allowing them to superovulate and extracting the oviduct force on the first day after mating.

 The genetically modified product (transgenic animal) is injected into the resulting fertilized egg by microinjection, etc., and artificially transplanted into the female oviduct and implanted to give birth. Can be obtained. In addition, after administration of luteinizing hormone-releasing hormone (LHRH) or its analogs to female animals, they were mated with animal males to produce pseudopregnant female animals with induced fertility. A method in which a fertilized egg is artificially transplanted and implanted in a pseudopregnant female animal may be used.

 [0017] The selection of pups or pups introduced with the above gene construct is performed by cutting the tip of the mouse or rat tail and extracting the polymer DNA (supervised by Tatsuji Nomura Genomic DNA is extracted by using a commercially available kit such as Motoya Katsuki, Kodansha (1987)) or DNAeasy Tissue Kit (manufactured by QIAGEN). The reporter gene in the VGAT expression control region can be confirmed by confirming the presence of DNA. In addition, when the reporter gene is a fluorescent protein gene, it can also be measured by measuring the amount of fluorescence emitted or observing with a fluorescence microscope or the like.

 [0018] The non-human mammal of the present invention is bred in an ordinary breeding environment after mating the individuals obtained as described above and confirming that the introduced DNA is stably maintained. The progeny can be obtained by this method, or the offspring can be obtained by repeating in vitro fertilization to maintain the strain.

As long as the non-human mammal of the present invention retains the above gene construct on the chromosome, other It may be an animal obtained by crossing with other animals.

 For example, since it is suggested that inhibitory-euron is involved in epilepsy, crossing the rat of the present invention with an epilepsy model animal such as an SHR rat allows observation of the inhibitory-euron dynamics in epilepsy, It is possible to obtain animals useful for analysis of the etiology of epilepsy and screening for therapeutic drugs.

 VGAT is also known to be expressed on splenic islets of Langernons. By crossing rats of this invention with diabetes model animals such as GK rats, Animals useful for observation, analysis of the etiology of diabetes, and screening of therapeutic agents can be obtained.

 The non-human gene-modified animal of the present invention, or the nerve tissue that can also obtain the animal power, is an inhibitor of GABA neurons and the like, electrophysiological analysis and morphology of GABA neurons using the amount of protein translated by the reporter gene as an index. It can be used for scientific analysis. It can also be used for the developmental analysis of GABA-euron, molecular biological analysis specific to GABA-euron, and in vivo expression dynamics analysis of VGAT gene.

 When a fluorescent protein gene is used as the reporter gene, the expression level of the fluorescent protein can be observed and measured as the fluorescence amount. The method for measuring the amount of fluorescence can be visualized by a fluorescence microscope or the like, quantified by FRET (Fluorescence Resonance Energy Transfer), the two-photon excitation method, or flow cytometry.

 Specifically, the inhibitory euron derived from the animal of the present invention is electrically or drug-stimulated, and this stimulation changes the fluorescence intensity in inhibitory neurons or synapses. By observing, functional or morphological changes of nerve cells or synapses to the stimulation can be analyzed. Such an analysis elucidates the mechanism of action and interaction of physiological pharmacological effects induced by drugs or stimuli on inhibitory neurons or synapses in the central nervous system. The knowledge gained from these analyzes will lead to the elucidation of the mechanisms of neurotransmission and pharmacological action.

In addition, since the VGAT gene is also expressed in glycine-euron, glycine-euron can also be visualized in vivo, and the functional analysis of glycine-euron and the pathogenesis of diseases involving glycine neurotransmission can be elucidated. Is available. [0020] Since the non-human genetically modified animal of the present invention can visualize inhibitory neurons or synapses in a living body, it can be used for screening and evaluation of neurotransmitters in nerve cells or synapses. it can. In the screening method of the present invention, the non-human mammal of the present invention is used to treat the animal with a test substance, analyze changes in nerve cells or synapses in the animal, and treat this with an untreated control animal. This can be done by comparing with

 Examples of the test substance include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc., and these substances are novel substances. It may be a known substance. Examples of a method for treating the animal with a test substance include known methods commonly used such as oral administration and intravenous injection, and can be appropriately selected according to the symptom of the test animal, the nature of the test substance, and the like. Good.

 In addition, the screening method of the present invention can also be performed using nerve tissue obtained from the genetically modified animal of the present invention.

 Examples of changes that appear in nerve cells or synapses include structural or morphological effects that accompany changes in the expression level of fluorescent proteins and the number of synapses.

 Example

 [0021] Gene construction

 (l) Selection of E. coli artificial chromosome (BAC) clones containing VGAT gene

 Three clones of E. coli artificial chromosome (BAC) clones containing the VGAT gene were identified by PCR from a mouse E. coli artificial chromosome (BAC) library (Genome system, St. Louis, MO, U.S.A.). By purchasing these 3 clones (# 44G06, # 160L22, # 184J1) from Genome system and determining the nucleotide sequence of the insertion site of each clone, the position of the VGAT gene in the insertion site of each clone is determined. Were determined. As a result, the # 160L22 clone has about 100 kb on the 5 ′ side of the VGAT gene, about 25 kb on the 3 ′ side, and has an insert of about 130 kb in total, including the VGAT gene (about 5 kb). found.

 [0022] (2) Insert venus gene into VGAT gene 'Preparation of modified BAC clone

Match the frame to the translation start codon (ATG) of exon 1 of the VGAT gene. The s gene (Nat Biotechnol. 2002 Jan; 20 (l): 87-90.) was inserted. In other words, it contains the Venus gene followed by the SV40poly (A) sequence, its 5-end is the VGAT gene 5-homology region (0.94 kb 5 '-expression control region), and the 3'-end is VGAT gene A DNA containing a 3'-homologous region (a region containing 1.39 kb of Ettason 1 and its downstream region) was prepared and used as a homologous recombination shuttle vector (pKOV—Kan; Lalioti and Heath, Nucleic Acids Res. 29 ( 3): E14. 2001), a targeting construct, VGAT-venus / KOV (Fig. 1) was prepared. VGAT-venus / KOV was introduced into # 160L2 2 clones, and by homologous recombination, VGAT-venus BAC clone with venus gene and poly A sequence inserted into ATG position of VGAT gene on # 160L22 clone was Lalioti and It was prepared according to the method of Heath (2001).

[0023] (3) Production of VGAT-venus rat

 BAC DNA was purified from the VGAT-venus BAC clone prepared as described above, and the DNA was injected into a rat fertilized egg and then transplanted into a pseudopregnant mouse to obtain 21 offspring (Founder). Whether or not the VGAT-venus gene was introduced was determined by PCR. The prime r used was designed in the venus gene, and the sequence is as follows.

 venus- F: 5,-ATGGTGAGCAAGGGCGAGGAGCTGT-3 (SEQ ID NO: 1)

 venus- R: 5,-TTACTTGTACAGCTCGTCCATGCCGA-3 '(SEQ ID NO: 2).

 As a result, it was found that of 21 pups, 2 were transgene rats (VGAT-venus rats) into which VGAT-venus gene was introduced. As a result of mating both with wild-type rats, the transgene was passed on to the next generation and established as a line.

[0024] (4) Analysis of VGAT-venus rat

 As a result of observing the VGAT-venus rats 1 to 3 days after birth with a fluorescence microscope, autofluorescence was observed on the head. Furthermore, as a result of observing the paraformaldehyde-fixed tissue with a fluorescence microscope, we observed strong autofluorescence in the brain (Fig. 2).

Whether or not cells with autofluorescence are inhibitory-euron Brain tissue specimens from 3-week-old VGAT-venus rats were prepared, observed for autofluorescence of GFP, and immune tissue using anti-GFP and anti-GABA antibodies匕 匕 匕 匕 examined. Figure 3 shows the results using cerebral cortex specimens. Anti-GFP antibody-positive cells are small to medium in size, have various morphologies, and have scattered GABA-euron characteristics. In addition, using GFP autofluorescence and anti-GABA antibody In addition, double staining revealed that GFP autofluorescent cells were anti-GABA antibody positive cells. Figure 4 shows the results using cerebellar cortex specimens. In the cells where GFP fluorescence was observed, a large number of small cells were observed in the molecular layer, and a small number of cells were scattered in the granular layer. The cells in which the fluorescence of GFP is observed are consistent with the distribution of stellate cells, sputum cells, and Golgi cells in the granule layer, known as GABA-euron. Anti-GFP antibody-positive cells are also consistent with the distribution of these GABA neurons. These results suggest that cells with GFP autofluorescence in the cerebral cortex and cerebellar cortex are GABA-uron, the main inhibitory-euron. Therefore, it was proved that VGAT-venus rat is useful for functional analysis of GABA-euron.

Industrial applicability

 The non-human mammal of the present invention is an inhibitory-euron that efficiently expresses a reporter gene. Therefore, by using the non-human mammal of the present invention, inhibitory-euron can be clearly identified and visualized under living conditions. In addition, electrophysiological experiments for analyzing the molecular mechanism of information transmission and pharmacological experiments for investigating the activity of various drugs acting on inhibitory neurons and their mechanism of action are much easier than before. Can be done. By using the nerve cells or synapses of the non-human mammal of the present invention, screening for therapeutic agents for cranial nerve diseases such as anxiety, depression, epilepsy, and nerve cell death can be performed efficiently.

Claims

The scope of the claims  [1] A non-human mammal having a reporter gene operably linked to a DNA fragment containing a 5 'expression regulatory region of about 10 lOOkbp of the vesicular GABA transporter gene on the chromosome.  [2] The non-human mammal according to claim 1, wherein the reporter gene is a gene further linked to the 3 ′ expression regulatory region of the vesicular GABA transporter gene. [3] A gene construct comprising a vesicular GABA transporter gene comprising a 5 'expression regulatory region of about lOOkbp and a reporter gene inserted at the position of the translation initiation codon of the gene is retained on the chromosome. The non-human mammal described in 1.  [4] The non-human mammal according to any one of claims 1 to 3, wherein the reporter gene is a Venus gene.  [5] The non-human mammal according to any one of claims 1 to 4, which is a rat. [6] A test substance is added to the non-human mammal according to any one of claims 1 to 5 or nerve tissue obtained from the animal, and the expression level of the reporter gene in inhibitory euron is used as an index. A screening method for a neurotransmission-regulating drug, comprising selecting a substance having an inhibitory activity to control neurotransmission of euron force.