JP5308651B2 - Drp1-deficient non-human mammal - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a model system effective for drug screening. <P>SOLUTION: The model animal that is effective for various diseases is a knockout non-human mammal, having a tissue-specifically deleted function of Drp1 gene. <P>COPYRIGHT: (C)2009,JPO&amp;INPIT

Description

  The present invention relates to a Drp1-deficient non-human mammal and use thereof.

  Mitochondria are organelles (intracellular organelles) surrounded by a double membrane that performs oxygen respiration, and play an indispensable function for energy production and, in turn, maintenance of life activity. On the other hand, oxidative stress, which is a secondary product of oxygen respiration in mitochondria, is known to give various damages to the living body, and its involvement in various pathological conditions and diseases is strongly suggested. Mitochondria have its own DNA separate from the nuclear genome, and it has been reported that the damage and accumulation of mutations can cause mitochondrial disease and that it can be a major cause of aging. Mitochondria is also known to play a central role in the process of inducing apoptosis by releasing various apoptosis-related proteins including cytochrome C into the cytoplasm, transitioning their membrane permeability, and determining cell death. .

  It is known that mitochondria maintain their functions while dynamically changing their structure in vivo, and greatly change their structure by tissue differentiation. In cultured cells, it is also known that mitochondria dynamically change their structure while repeating fusion and division. However, the physiological significance of such mitochondrial dynamics has not been fully elucidated.

  Drp1 (Dynamin-related protein 1), a dynamin-like GTPase, is a protein involved in the control of mitochondrial division. Drp1 is mainly localized in the cytoplasm, but is also frequently observed at the mitochondrial fission point. When the function of Drp1 is suppressed, mitochondrial division is inhibited and very long mitochondria are formed (Non-patent Documents 1 and 2). It has also been reported that mitochondrial morphology is controlled by the balance between Mitofusin protein-dependent fusion and Drp1-dependent division (Non-patent Document 3). That is, when mitochondria fusion is inhibited, only division proceeds to form short fragmented mitochondria, and conversely, inhibition of division forms long connected mitochondria (Non-patent Document 2). However, little is known about the effects of Drp1 mutations on mitochondrial function at the individual or tissue level.

  By the way, at present, model animals for various diseases have been produced and used effectively for the development of new drugs. However, in the case of diseases with complicated onset causes, it is quite difficult to create a model animal that faithfully mimics the pathology. In many model animals, the pathology shown is more extreme or peculiar than that of an actual patient, or only a few symptoms are shown.

  For example, various model animals for diabetes have been prepared. Diabetes is at the core of the rapidly increasing lifestyle-related diseases, and the development of preventive and therapeutic methods is urgently needed to realize a healthy aging society. The onset of diabetes is explained by two pathological conditions, insulin secretion failure (insulin deficiency) and insulin resistance (insulin action disorder). Insulin is a hormone that has the effect of lowering blood sugar level by absorbing sugar in the blood to the cell. When the action is reduced, the blood sugar level increases, but the sugar in the cell is insufficient. If this condition persists, the cells will not be able to sustain vital activities, resulting in various organ damage such as blindness and renal failure.

  Type 1 diabetes is one of the autoimmune diseases and is characterized by an absolute deficiency of insulin because insulin is depleted from the destruction of pancreatic β cells. Therefore, supplementation with insulin injection is indispensable for the treatment. A model mouse for type 1 diabetes is known, and research on the treatment of type 1 diabetes using the model mouse is also progressing (Non-patent Document 4).

  On the other hand, type 2 diabetes, which accounts for the majority of diabetic patients and is rapidly increasing as a lifestyle-related disease, is also called non-insulin-dependent diabetes from a clinical point of view. It becomes an exacerbation factor. Whether insulin secretion deficiency or insulin resistance is strongly related varies depending on individual cases and courses, but compared with Westerners, Japanese insulin occupies a large part of the cause. In normal cases, glucose is absorbed after meals, and when blood sugar levels begin to rise, insulin is secreted instantaneously (glucose-responsive insulin secretion). In the case of insulin secretion deficiency, this response is lacking. Insulin is secreted later than the increase in blood sugar, and postprandial hyperglycemia is characteristic. Furthermore, if this state continues for a long time, it is often clinically experienced that pancreatic β-cells are exhausted from the load of insulin secretion, resulting in an absolute deficiency of insulin secretion as in type 1 diabetes. However, the molecular mechanism of pancreatic β-cell exhaustion is not clear. Unlike type 1 diabetes, type 2 diabetes has a complex etiology, and development of model animals that sufficiently mimic human pathology has not progressed. The production of type 2 diabetes model animals has also been reported (for example, Patent Document 1), but the type 2 diabetes model animals reported so far are extremely obese, overeating, excessive leptin concentration or lack of leptin, high Exhibiting extreme symptoms such as insulinemia.

  On the other hand, model animals for neurodegenerative diseases have great expectations for new drug development. Neurodegenerative diseases such as senile dementia, cerebrovascular dementia, Alzheimer's disease, amyotrophic lateral sclerosis, and Parkinson's disease have different etiologies (vascular, metabolic, infectious, etc.), but neuropathy is a common pathological condition. It presents memory and cognitive impairment due to cell and neural network failure. There have been many reports of neurodegenerative disease model mice so far. A model animal to be imitated has not been produced (for example, Patent Document 2).

JP 2005-295867 A Special table 2003-504015 gazette Smirnova E, et al., J Cell Biol. (1998) Oct 19; 143 (2): 351-8. Naotada Ishihara, Katsuyoshi Mihara, “Control Mechanism of Mitochondrial Fission and Fusion”, Cell Engineering Vol.24, No.8 (2005), Shujunsha, p.799-803 Ishihara N. et al., Biochem. Biophys. Res. Comm. 301 (2003) p.891-898 Kodama et al., Science (2003) Nov. 14; 302 (5648): 1223-1227

  An object of the present invention is to provide a model system effective for drug screening.

  As a result of intensive studies to solve the above problems, the present inventors have found that a knockout non-human mammal deficient in the function of the Drp1 gene in a tissue-specific manner can be a good model animal for various diseases. The present invention has been completed.

That is, the present invention includes the following.
[1] A knockout non-human mammal in which the function of the Drp1 gene is deficient in a tissue-specific manner.
In this knockout non-human mammal, the function of the Drp1 gene is preferably deficient by deletion, substitution and / or insertion of at least a part of the base sequence on the Drp1 gene or its expression control region.
The knockout non-human mammal is more preferably a rodent.
The knockout non-human mammal can be used as a glucose-responsive insulin secretion deficient model animal when the function of the Drp1 gene is deficient specifically in pancreatic β cells.
The knockout non-human mammal can be used as a model animal for synapse formation failure when the function of the Drp1 gene is specifically deficient in neurons.

[2] A test substance is administered to the knockout non-human mammal in which the function of Drp1 gene is specifically deficient in pancreatic β cells or the pancreatic β cell in which the function of Drp1 gene obtained therefrom is deficient A method for screening a glucose-responsive insulin secretion improving agent.

[3] A test substance is administered to the knockout non-human mammal deficient in nerve cell-specific functions of Drp1 gene or a nerve cell deficient in the function of Drp1 gene obtained therefrom. And screening method for synapse formation promoter.

  The knockout non-human mammal deficient in tissue-specific Drp1 gene function according to the present invention has various disease states associated with mitochondrial dysfunction, such as glucose-responsive insulin secretion dysfunction and synaptogenesis phenotype. Show. Therefore, the drug screening method using this knockout non-human mammal or a cell deficient in the function of Drp1 gene obtained from it has failed to detect glucose-responsive insulin secretion failure, which has not been previously known to be associated with mitochondrial dysfunction. It is useful in searching for an effective drug for the improvement of the accompanying type 2 diabetes and its complications, or neurodegenerative diseases accompanied by synapse formation failure.

Hereinafter, the present invention will be described in detail.
1. Drp1 gene deficient (knockout) non-human mammal The present invention relates to a knockout non-human mammal in which the function of the Drp1 gene on the chromosome is tissue-specifically deficient.
The “Drp1 gene” according to the present invention means a gene encoding Drp1 (Dynamin-related protein 1) protein which is a dynamin-like GTPase of mammals. Drp1 is known as a protein mainly localized in the cytoplasm, and is also called by other names such as Dynamin-1 like (DNM1L), Dnm1, Dlp1, DVLP, and dymple in mammals. Drp1's yeast homologue, Dnm1 (Dnm1p), is undergoing considerable research. The nematode homologue of Drp1 is called DRP-1, and it varies depending on the species in plants, but the Drp1 homologue of Arabidopsis is called ADL2b. The “Drp1 gene” referred to in the present invention may include the mRNA and cDNA thereof in addition to the genomic DNA encoding the Drp1 protein.

  The nucleotide sequence and amino acid sequence of the Drp1 gene are known in various animals such as Drosophila, yeast, nematode, Arabidopsis thaliana, mouse, rat, and human. The base sequence (cDNA sequence or genomic sequence) of the Drp1 gene can be easily obtained through public databases (including international base sequence databases) such as DDBJ, EMBL, GenBank, and various commercial databases. For example, as the sequence of the Drp1 gene, mouse: GenBank accession numbers NM_152816, NM_001025947, human: GenBank accession numbers NM_012062, NM_012063, NM_005690, rat; NM_053655 and the like have been reported. For non-human mammals whose Drp1 gene sequence is not registered in public or commercial databases, the base sequence of the non-human mammal's endogenous Drp1 gene is cloned based on homology with the known Drp1 gene. Can be determined. For example, a genomic DNA library or cDNA library of the animal is prepared, and a known Drp1 gene or a part thereof derived from a genetically close species, or a highly conserved region between species in the Drp1 gene By screening the library using it as a probe, the Drp1 gene of the target animal can be identified and sequenced.

  For example, the mouse Drp1 gene is mapped on mouse chromosome 16. Detailed gene information of the mouse Drp1 gene is based on Gene ID: 74006 from the database of NCBI (accessible from National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/) or MGI It can be obtained from the database of (Mouse Genome Informatics; accessible from http://www.informatics.jax.org/) based on the name “dynamin-1 like”.

  In the present invention, “the function of the Drp1 gene is tissue-specifically deficient” means that the Drp1 gene on the chromosome is genetically modified so that it is not normally expressed in a specific tissue. Means that Not only when the Drp1 gene product is not expressed at all, but also when the gene product is expressed, if it does not have a normal function or does not achieve a sufficient expression level, “Drp1 gene function is deficient. It will be. The state in which the Drp1 gene is not normally expressed in this way is the modification of at least part of the base sequence of the Drp1 gene on the genome, or at least part of the expression control region such as the transcriptional regulatory region or promoter region of the Drp1 gene. It can be caused by modification of the base sequence, for example, deletion, substitution, and / or insertion (ie, insertion of another base sequence). A typical example of an animal deficient in the function of the Drp1 gene is an animal deficient in all or part of the coding (exon) region of the Drp1 gene.

  The site where the deletion, substitution or insertion is performed and the nucleotide sequence to be deleted, substituted or inserted are not particularly limited as long as the normal function of the Drp1 gene is lost. However, a mutation that deletes at least a part of the coding region of the Drp1 gene (for example, a region containing one or more exons) can more reliably cause the function of the Drp1 gene to be lost. Usually, a region containing at least one exon of the Drp1 gene, for example, a region containing exon 2 may be deleted. The method for deleting the function of the Drp1 gene will be described in the next section.

  In the present invention, “tissue-specific deficiency” means that the deficiency is specifically caused only in a specific tissue or mainly in the specific tissue. For example, in addition to a pancreatic β-cell specific defect, a case where a defect in the hypothalamus is also recognized as secondary is included in “a pancreatic β-cell specific defect”. In the present invention, the term “tissue-specific” “tissue” includes not only tissues based on histological classification (eg, connective tissue, epithelial tissue, nerve tissue, muscle tissue, etc.) but also organs (organs) and cells. It also includes any in vivo portion such as a species, cell population, etc. In the Drp1 gene-deficient non-human mammal of the present invention, the type of such tissue (eg, organ or cell) that specifically lacks the function of Drp1 gene is not particularly limited, and any organ or cell type in the living body Preferred examples include pancreatic β cells and nerve cells. Non-human mammals whose Drp1 gene function is tissue-specifically deficient are useful as model animals for various diseases, for example, non-human mammals whose Drp1 gene function is specifically deficient in pancreatic β cells or neurons The animals are particularly useful as disease models of glucose-responsive insulin secretion failure and synapse formation failure, respectively.

  The Drp1 gene-deficient non-human mammal of the present invention is able to detect alleles on both chromosomes even if the function of the Drp1 gene is deficient for alleles on one chromosome in the cell (heterozygote). It may be deficient (homozygote), and it may vary from cell to cell.

  In the present invention, the “knockout non-human mammal” refers to a non-human mammal having at least one tissue (for example, a specific organ or cell type) that lacks the function of the gene on the chromosome due to genetic modification. The “non-human mammal” according to the present invention is not particularly limited as long as it is a mammal other than a human, but is preferably a rodent such as a mouse, a rat, a guinea pig, or a rabbit. Most preferred is a mouse that can be easily performed.

2. Method for producing Drp1 gene-deficient non-human mammal The Drp1 gene-deficient non-human mammal of the present invention specifically lacks the function of Drp1 gene in a target tissue (eg, target cell type) by a known genetic engineering technique. Can be produced. Such tissue-specific gene modification techniques include, but are not limited to, the Cre-loxP system (eg Gu, H., et al., Science, (1994) 265: 103-106, Kuhn R et al., Science, 269, 1427-1429, 1995) or FLP-frt systems (eg Dymecki, SM, Proc. Natl. Acad. Sci. USA, (1996) 93: p.6191-6196, Rodriguez, CI , et al., Nat. Genet., (2000) 25: 139-140) or combinations thereof (eg Meyers, EN et al., Nat. Genet., (1998) 18: 136-141) A targeting method can be preferably used. For details of the experiment on the conditional targeting method, refer to Chapter 7 of the “Genetic Engineering Handbook” (Yodosha) edited by Muramatsu et al. The production of conditional knockout non-human animals that cause tissue-specific deletion of the target gene used in the conditional targeting method is also common in "Experimental Medicine Separate Edition 4th Edition New Genetic Engineering Handbook" (Yodosha) etc. Although it can be carried out by a routine method according to an experimental document, such a conditional knockout non-human animal has already been reported to produce many kinds of animals, and can be purchased from The Jackson laboratory or the like. Detailed information on conditional knockout non-human animals that cause tissue-specific defects in existing target genes, such as http://www.mshri.on.ca/nagy/ and http://www.jax.org/ You can also get it from

  The loxP (locus of X-ing-over) sequence is a DNA sequence (5'-ATAACTTCGTATAGCATACATTATACGAAGTTAT-3 ') consisting of 34 base pairs and a recognition sequence for Cre (Causes recombination) recombination enzyme. The two loxP sequences on the gene cause specific recombination in the presence of the Cre protein, deleting the region flanked by loxP sequences or causing its inversion. If you replace the genomic region you want to delete with the loxP sequence in the same direction, and place the Cre gene under the control of a cell (tissue) -specific promoter in the cell, the cell that expresses the promoter ( By expressing the Cre protein only in the tissue), the genomic region sandwiched between the loxP sequences can be deleted.

  In the following, this Cre-loxP system will be described as an example.

1) Construction of targeting vector First, a targeting vector used for deleting the function of the Drp1 gene is constructed. Prior to the construction of the targeting vector, a genomic DNA library of the animal to be used is generally obtained or prepared. As this genomic DNA library, it is preferable to use a genomic DNA library prepared from an animal strain from which the ES cells to be used are derived so that the recombination frequency does not decrease due to polymorphism or the like. The prepared genomic library is screened using Drp1 cDNA or a partial sequence thereof as a probe to obtain a clone containing the genomic DNA sequence of Drp1 gene. Further, a restriction enzyme map showing the position of each exon and the restriction enzyme site is prepared for the obtained genomic DNA clone using sequencing, Southern blot analysis, restriction enzyme digestion and the like. When using genomic DNA clones for which information such as restriction enzyme maps have already been published, the known information may be used directly. Based on this restriction enzyme map, the region to be deleted from the Drp1 gene is determined, and a targeting vector is designed. The region to be deleted is not limited, but a region containing at least one exon is preferable. In general, a genomic region adjacent to the 5 ′ side of the deletion region and a genomic region adjacent to the 3 ′ side are selected as homologous regions, and DNA fragments corresponding to these regions are prepared. Then, in an appropriate vector or gene cassette, a genomic region adjacent to the 5 ′ side of the region to be deleted, a loxP sequence, a region to be deleted, a loxP sequence, a marker gene or a reporter gene (may include a promoter or the like), By inserting the loxP sequence and the genomic region adjacent to the 3 ′ side of the region to be deleted in this order in the 5 ′ → 3 ′ direction, a targeting vector can be prepared. All loxP sequences must be placed in the same direction.

  Examples of marker genes include neomycin resistance genes (gene cassettes such as pGKneo and pMC1neo), positive selection markers such as hygromycin B phosphotransferase gene, herpes simplex virus thymidine kinase gene (HSV-TK), diphtheria toxin A fragment (DT -A) and other negative selectable marker genes can be mentioned, but not limited thereto. Examples of reporter genes include green fluorescent gene (GFP), β-galactosidase gene (LacZ), luciferase gene (luc), and the like. The vector can be constructed using genetic engineering techniques such as PCR and ligation known to those skilled in the art.

2) Introduction of targeting vector into ES cell Next, the constructed targeting vector is introduced into a totipotent cell such as embryonic stem cell (ES cell). ES cells have been established in mice, hamsters, pigs, etc., especially in mice, K14 strain, E14 strain, D3 strain, AB-1 strain, J1 strain, R1 strain, TT2 derived from 129 mice Various cell lines, such as strains, are available. In mice, embryonic carcinoma cells (EC cells) can be used instead of ES cells.

  Prior to the introduction of the targeting vector, ES cells are cultured in an appropriate medium. For example, in the case of mouse ES cells, mouse fibroblasts or the like may be used as feeder cells, and a liquid medium for ES cells (for example, manufactured by GIBCO) may be added thereto for co-culture.

  Introduction of a targeting vector into ES cells can be carried out by a known gene introduction method such as electroporation, microinjection, calcium phosphate method and the like. ES cells into which a targeting vector has been introduced can be easily selected by expressing a marker gene or reporter gene inserted into the vector. For example, in the case of a cell into which a neomycin resistance gene has been introduced as a marker gene, primary selection can be performed by culturing in a medium for ES cells supplemented with G418.

  In ES cells into which a targeting vector has been introduced, a portion of the Drp1 gene on the chromosome is replaced by the vector by homologous recombination, and the loxP sequence is located on both sides of the endogenous Drp1 gene or the deletion site in its regulatory region. And a marker gene or reporter gene and a loxP sequence are preferably introduced downstream thereof. At this point, no deletion has occurred in the Drpl gene. Whether or not the desired homologous recombination has been achieved can be determined by genotype analysis using Southern blotting, PCR method or the like. Genotype analysis by PCR can be carried out by detecting specific amplification products of wild type and mutant Drp1 gene, respectively. The ES cells into which the targeting vector has been appropriately introduced in this way are further cultured for the next stage.

3) Production of chimeric animals ES cells (recombinant ES cells) into which a targeting vector has been introduced are introduced into early embryos derived from another strain that is apparently different from the strain from which the ES cells are derived. It is preferable to generate as. For example, in the case of a mouse, a C57BL / 6 mouse having a black hair color and various gene loci that can be used as markers are different from those of a 129 mouse for ES cells derived from the 129 line having an albino hair color, etc. It is desirable to use early embryos. Thereby, the chimera mouse can judge the chimera rate from the hair color.

  ES cells are introduced into early embryos by the aggregation method (Andra, N. et al. Proc. Natl. Acad. Sci. USA, 90, 8424-8428, 1993, Stephen, AW et al. Proc. Natl. Acad. Sci. USA, 90, 4582-4585, 1993), microinjection method (Hogan, B. et al. “Manipulating the Mouse Embryo” Cold Spring Habor Laboratory Press, 1988).

  In the aggregation method, an ES cell mass is inserted and cultured between two 8-cell embryos or morulas from which the zona pellucida has been removed, and a chimeric embryo is obtained by aggregation. If this chimeric embryo is transplanted into the uterus of a foster parent (pseudopregnant animal) and generated, a chimeric animal can be obtained.

  The microinjection method is a method in which ES cells are directly injected into a blastocyst. That is, a recombinant embryonic stem cell is directly injected under a microscope into a blastocyst collected from an animal using a micromanipulator or the like to produce a chimeric embryo. If this chimeric embryo is transplanted into the uterus of a foster parent (pseudopregnant animal) and generated, a desired chimeric animal can be obtained.

4) Preparation of Drp1 gene-deficient non-human mammal A chimeric animal obtained from a foster parent is further backcrossed to a wild-type animal (eg, a C57BL / 6 mouse if it is a mouse). Among the obtained animals, there should be an individual heterozygous for the Drp1 gene into which the loxP sequence has been inserted by homologous recombination with the targeting vector. The genotype of each individual can be determined primarily by appearance characteristics such as hair color, and can be determined by genotype analysis using the Southern blotting or PCR method described above. Next, the heterozygous recombinant non-human mammals thus obtained can be mated to obtain an animal having a homozygous Drp1 gene into which the loxP sequence has been inserted.

  In order to obtain a non-human mammal (for example, mouse) in which the Drp1 gene has been disrupted in a tissue-specific manner, an animal having the Drp1 gene into which the loxP sequence has been inserted homozygously obtained in the above-described manner is used in a tissue-specific Cre protein. Transgenic non-human animals that have the Drp1 gene inserted with the loxP sequence heterozygously and have the Cre transgene (a foreign gene encoding Cre protein) are obtained by mating be able to. Furthermore, about half of the pups obtained by mating an animal having a homozygous Drp1 gene having the loxP sequence inserted into this animal is a conditional knockout animal in which the Drp1 gene is specifically disrupted in the target tissue. The animal thus prepared is the tissue-specific conditional Drp1 gene-deficient non-human mammal of the present invention.

3. Use of Drp1 gene-deficient non-human mammal as a model animal In the Drp1 gene-deficient non-human mammal of the present invention, the intracellular mitochondrial division is inhibited in a tissue-specific manner in which the function of Drp1 gene is deficient. Changes specific to the shape occur. Such a tissue-specific Drp1 gene-deficient non-human mammal can be used as a good model animal for various diseases associated with inhibition of mitochondrial division. Examples of non-human mammals deficient in the Drp1 gene specifically for pancreatic β cells or nerve cells are shown below.

1) Pancreatic β cells of a non-human mammal deficient in the pancreatic β-cell-specific Drp1 gene deficient in glucose-responsive insulin secretion model animals , mitochondrial division is suppressed, mitochondria are lengthened, and mitochondria accumulate around the nucleus in the cell The tendency to do is observed. In this non-human mammal deficient in the pancreatic β cell-specific Drp1 gene, the pancreatic islets of Langerhans are normally constructed and insulin synthesis is also performed in pancreatic β cells, but glucose responsiveness is a characteristic pathology of type 2 diabetes. Insulin secretion is impaired, and glucose tolerance decreases rapidly. This pancreatic β-cell-specific Drp1 gene-deficient non-human mammal exhibits postprandial (after glucose load) hyperglycemia as a typical symptom of glucose-responsive insulin secretion failure. On the other hand, in non-human mammals deficient in pancreatic β cell-specific Drp1 gene, preferably, fasting blood glucose does not increase even when compared with the wild type, and no significant increase in body weight is observed.

  Glucose tolerance and insulin secretion ability can be measured, for example, as follows. First, after fasting (for example, 16 hours) a test animal (for example, an 8-week-old mouse), venous blood is collected (from the tail vein for mice and rats), and its blood glucose level and serum insulin level are measured in advance. Keep it. Subsequently, glucose (for example, 2 g / kg body weight) is administered into the abdominal cavity of a test animal fasted, blood is collected at a predetermined time (preferably over time) after glucose administration, and blood glucose level and blood insulin level are measured. taking measurement. Blood collection may be performed at an arbitrary time point, and for example, it may be performed at intervals of 15 minutes at the beginning and at intervals of 30 minutes in the latter half from about 0 minutes to 3 hours after glucose administration. In the control test animals (wild type), the blood glucose level rises rapidly after the time when blood glucose level begins to drop (after about 30 minutes in the mouse), and then the high blood glucose level is maintained even after 120 minutes in the mouse, for example. If it is determined that the blood glucose processing capacity is reduced, it is determined that the glucose tolerance is reduced. In addition to such post-glucose hyperglycemia, if the blood insulin level (initial secretory volume) is significantly low in about 15 minutes in the mouse, the test animal has a glucose-responsive insulin secretion deficiency. It is judged to show.

  Furthermore, for pancreatic β cell-specific Drp1 gene-deficient non-human mammals, pancreas slices were prepared, immunohistologically stained using, for example, an anti-insulin antibody, and the area of pancreatic islets of Langerhans was measured to obtain control mice ( It is possible to examine whether or not insulin synthesis in the pancreas of the test animal is normal by comparing with a pancreatic section of (wild-type phenotype).

  Pancreatic β-cell-specific Drp1 gene-deficient non-human mammals can be used as model animals for glucose-responsive insulin secretion failure, but they mimic the pathology of type 2 diabetes very well. It can be used as a model animal. Furthermore, the pancreatic β cell-specific Drp1 gene-deficient non-human mammal can also be used as a model animal for type 2 diabetes complications accompanied by glucose-responsive insulin secretion failure.

  Therefore, the pancreatic β cell-specific Drp1 gene-deficient non-human mammal of the present invention, or the pancreatic β cell deficient in the function of the Drp1 gene obtained from the animal, is a glucose-responsive insulin secretion deficiency model animal or model cell, respectively. It can be used for screening for a drug that promotes insulin secretion (glucose-responsive insulin secretion improving agent) for alleviating glucose-responsive insulin secretion failure. Accordingly, the present invention provides a glucose-responsive insulin characterized by administering a test substance to such a pancreatic β-cell-specific Drp1 gene-deficient non-human mammal or a pancreatic β-cell deficient in the function of the Drp1 gene obtained therefrom. A screening method for secretagogues is also provided. More specifically, a test substance that promotes insulin secretion as a result of administering a test substance to the pancreatic β cell-specific Drp1 gene-deficient non-human mammal or the pancreatic β cell is selected as a glucose-responsive insulin secretion improving agent. can do. Administration of the test substance to the animal may be oral administration or parenteral administration (intravenous injection, subcutaneous injection, intraperitoneal injection, rectal administration, etc.) according to the intended administration route of the test substance to be used. May be. Administration of the test substance to the cells may be performed by adding the test substance to the culture medium of pancreatic β cells.

  This test substance may be any substance. Examples of the test substance include nucleic acids including DNA and RNA, proteins including enzymes, antibodies, peptides, small molecule organic or inorganic compounds.

  The glucose-responsive insulin secretion improving agent thus obtained can be a candidate therapeutic for type 2 diabetes or its complications.

2) Synergic hypoplasia model animal nerve cell-specific Drp1 gene-deficient non-human mammal nerve cells have enlarged mitochondria and increased apoptosis. In a non-human mammal deficient in nerve cell-specific Drp1 gene, synapse formation of nerve cells is observed in the brain. In the present invention, “synapse formation failure” refers to a state where synapses are not sufficiently formed. “Synapse formation failure” is not limited to the following, but the synapse formation process is not started or stopped in the middle, the synapse formation process does not proceed normally, or the formed synapse is killed or dropped out. It can be caused by decreasing etc. “Synapse formation failure” in the present invention is typically indicated by a decrease in the number of synapses detected in a non-human mammal deficient in a nerve cell-specific Drp1 gene compared to a normal animal as a control.

  The failure of synapse formation in Drp1 gene-deficient neurons can be examined, for example, as follows. First, the Drp1 gene-deficient non-human mammal ES cells are cultured on a suspension culture dish (for about 2 days) using a medium (for example, LIF (-)) to produce an embryoid body. Culture in a cell differentiation induction medium. Examples of such a medium include DMEM / F12 medium supplemented with insulin, transferrin, selenium chloride, and human plasma fibronectin. Further, the cell mass is peeled from the culture vessel with trypsin or the like, and cultured in an appropriate medium such as DMEM / F12 medium supplemented with N2 supplement, B27 supplement, and human basic FGF (for example, 6 days). By doing so, neuronal cells having axons can be induced to differentiate in the case of wild type ES cells. Then, nerve cells and mitochondria may then be stained and observed using nerve cell specific antibodies or mitochondrial specific antibodies. As a result, if the presence of neuron-like cells is detected and these cells are shown to form a central nervous system-like network, the induced neuron is judged to have synapse-forming ability. The On the other hand, if neuron-like cells are detected but sufficient axon formation from the neuron body and central nervous system-like network formation are not observed, the neuron has sufficient synapse-forming ability. It is judged that there is no synapse formation dysfunction. Such nerve cell-specific Drp1 gene-deficient non-human mammals can be used as model animals for synapse formation failure, but synapse formation causes disruption of neuronal circuit and induces general neurodegenerative pathology Therefore, it can be used as a model animal for neurological diseases such as neurodegenerative diseases and dementia.

  There are many reports on the relationship between synapse dysfunction and neurological diseases such as neurodegenerative diseases and dementia. For example, it has been reported that synapse reduction was significantly observed in the hippocampus of patients with Alzheimer's syndrome and moderate dementia (Scheff SW et al., Neurology, 2007 May 1; 68 (18): 1501-1508 ). In recent years, treatment of neurological diseases by holding synapses rather than nerve cells has been considered promising. For example, transplantation of bone marrow-derived mesenchymal hepatocytes into a Niemann-Pick disease type C model mouse that causes neuronal degeneration and exhibits progressive neurological symptoms promotes synapse formation and performs functional synaptic transmission. It has been reported that it was able to promote the formation of neural networks (Bae JS et al., Stem Cells., 2007 May; 25 (5): 1307-1316).

  Therefore, the nerve cell-specific Drp1 gene-deficient non-human mammal of the present invention can be used as a model animal for synapse formation failure to screen for a drug that promotes synapse formation (synapse formation promoter). Therefore, the present invention provides a screening method for a synapse formation promoter, characterized by administering a test substance to such a nerve cell-specific Drp1 gene-deficient non-human mammal or a nerve cell deficient in the function of the Drp1 gene obtained therefrom. Also provide. More specifically, a test substance is administered to the nerve cell-specific Drp1 gene-deficient non-human mammal or the nerve cell (for example, undifferentiated nerve cell derived from fetal brain) to induce nerve differentiation (for example, axon What promotes (elongation) can be selected as a synapse formation promoter. Administration of the test substance to the animal may be oral administration or parenteral administration (intravenous injection, subcutaneous injection, intraperitoneal injection, rectal administration, etc.) according to the intended administration route of the test substance to be used. May be. Administration of the test substance to the cells may be performed by adding the test substance to the culture medium of nerve cells.

  This test substance may be any substance. Examples of the test substance include nucleic acids including DNA and RNA, proteins including enzymes, antibodies, peptides, small molecule organic or inorganic compounds.

  The synapse formation promoter thus obtained can be a candidate therapeutic agent for neurological diseases such as neurodegenerative diseases and dementia.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.

[Example 1] Preparation of mice and cells knocked out of Drp1 gene Drp1 gene conditional knockout mice using the Cre-loxP system were prepared according to the following procedure (Figs. 1A and B).
First, in order to prepare a targeting vector, a mouse 129 / SV genomic library (STRATAGENE) was used as a probe and a 549 bp cDNA fragment of mouse Drp1 (the following primers: 5'-TCGAGTCCCCATTCATTGCAG-3 'and 5'-GGTCATTCCCGGTAAATCCAC-3' And a genomic DNA clone containing the mouse Drp1 gene was identified. A restriction enzyme map was prepared for the obtained clone, and based on this, a HindIII-XbaI fragment (approximately 3.6 kb) containing the upstream sequence of exon 2 of the Drp1 gene from the genomic DNA clone, and an XbaI-SpeI fragment containing exon 2 of the Drp1 gene A SpeI-EcoRV fragment (about 5.6 kb) containing exons 3 and 4 downstream of exon 2 of the Drp1 gene (about 1.8 kb) was excised. The HindIII-XbaI fragment (approximately 3.6 kb), loxP sequence (5′-ATAACTTCGTATAGCATACATTATACGAAGTTAT-3) in a pflox plasmid (a plasmid containing a neomycin resistance gene (neo) and two loxP sequences flanked on both sides) '), By inserting XbaI-SpeI fragment (about 1.8 kb), loxP sequence, neomycin resistance gene (neo), loxP sequence, SpeI-EcoRV fragment (about 5.6 kb) in the order of 5' → 3 ' A targeting vector was prepared. All loxP sequences were arranged in the same direction.

This targeting vector was introduced into mouse ES cells (J1 cell line; Li E. et al. Cell 69, 915-926, 1992) using an electroporation method, and then a medium containing 200 μg / ml G418. By ES cell selection, ES cells that were homologous recombinants were obtained. In this homologous recombinant ES cell (Drp1 ^{lox / +} ES cell), exon 2 of the Drp1 gene on the genome is replaced with exon 2 flanked by loxP sequences on both sides, and neomycin resistance gene and loxP are further downstream It has a Drp1 gene mutant into which the sequence has been introduced (FIG. 1A, middle panel).

Next, this homologous recombinant ES cell was injected into a blastocyst collected from a C57BL6 strain mouse, and transplanted into the uterus of a pseudopregnant mouse according to a conventional method to give birth to a chimeric mouse. Got. This chimeric mouse was backcrossed with a mouse of C57BL6 strain to obtain a pup mouse, thereby producing a mouse having a heterozygous Drp1 gene into which a loxP sequence was inserted (referred to as a lox hetero mouse or a Drp1 ^{lox / +} mouse). . Furthermore, a mouse having a homozygous Drp1 gene into which a loxP sequence was inserted (referred to as a lox homo mouse or a Drp1 ^{lox / lox} mouse) was prepared by crossing the heterozygous mice.

EIIa-Cre transgenic mice that express Cre protein in gametes (Lakso M et al, PNAS 93, 5860-5865, 1996) were used to produce Drp1 whole body-deficient mice. In EIIa-Cre transgenic mice, the expression of Cre protein (Cre recombinase) is controlled on the genome by the adenovirus EIIa promoter, and in particular, the expression of Cre protein is widely observed in gametes and early embryos. Here, EIIa Cre transgenic mice were purchased from The Jackson laboratory (Bar Harbor, ME) and used (Stock Number: 003724). First, by crossing the Drp1 ^{lox / +} mouse obtained above with this Ella-Cre transgenic mouse, a heterozygous knockout mouse (Drp1 ^+/- having a Drp1 gene disrupted by exon 2 deletion heterozygously) Mouse). Further, a homo knockout mouse (Drp1 ^{− / −} mouse) having the disrupted Drp1 gene in a homozygote was prepared by mating the Drp1 ^+/− mice.

Furthermore, in order to obtain mice in which the Drp1 gene was disrupted in a tissue-specific manner, nestin-Cre transgenic mice that express Cre protein in a nerve-specific manner and RIP-Cre transgenic mice that express Cre protein in a pancreatic β-cell-specific manner Were used respectively. The nestin-Cre transgenic mouse was created by Klein R. et al. and reported in Nat Genet 23: 99-103, 1999, and the expression of Cre protein is expressed in the genome by the rat nestin gene promoter and enhancer. It is cell-specifically controlled. In this example, nestin-Cre transgenic mice were purchased from The Jackson laboratory (Bar Harbor, ME) and used (Stock Number: 003771). On the other hand, RIP-Cre transgenic mice were produced by Magnuson M. et al. And reported in J Biol Chem 274: 305-315, 1999, and the expression of Cre protein controlled by the rat insulin II promoter was almost the same. Although it is found specifically in pancreatic β cells, very little expression is also seen in the hypothalamus. In this example, RIP-Cre transgenic mice were purchased from The Jackson laboratory (Bar Harbor, ME) and used (Stock Number: 003573). The Drp1 ^{lox / lox} mouse obtained above is crossed with a nestin-Cre transgenic mouse or a RIP-Cre transgenic mouse to produce a Cre transgene (ie, Cre-expressed in a pancreatic β cell-specific manner). A Drp1 ^{lox / +} mouse having a foreign gene encoding a protein) is obtained as a offspring mouse, and the Drp1 ^{lox / +} mouse is further crossed with a Drp1 ^{lox / lox} mouse, so that the Drp1 gene is nerve-specific or pancreatic β, respectively. We were able to create knockout (KO) mice that were specifically disrupted by cells (Drp1 ^{lox / lox} ; nestin-Cre mouse and Drp1 ^{lox / lox} ; RIP-Cre mouse, respectively).

Furthermore, fibroblasts were collected from Drp1 ^{lox / lox} mouse embryos, and a mouse fetal fibroblast cell line (Drp1 ^{lox / lox} MEF cells) having the Drp1 gene inserted with the loxP sequence in a homozygote was prepared. On the other hand, by culturing Drp1 ^{lox / +} ES cells in a selective medium containing a high concentration (4 mg / ml) of G418, ES cells (Drp1 ^{lox / lox} ES with homozygous Drp1 gene inserted with loxP sequence) Cell). Subsequently, the Cre gene is introduced into a Drp1 ^{lox / lox} MEF cell or a Drp1 ^{lox / lox} ES cell by exogenous introduction of the Cre gene using adenovirus, and the Cre protein is expressed to have a homozygous Drp1 gene disrupted (knocked out). Drp1 ^{− / −} MEF cells or Drp1 ^{− / −} ES cells were prepared.

For the Drp1 knockout mice and knockout cells prepared as described above, PCR amplification using the genomic DNA extracted by the conventional method as a template and the primer 1F and 2R set and the primer 1F and 3R set shown in FIG. 1A is performed. Then, from the size of the amplified fragment, the presence of the loxP sequence and the insertion of the loxP sequence and the neo gene was confirmed. The sequences of the primers used and the sizes of the amplified fragments are as follows.
・ Primer 1F: 5'- CAGCTGCACTGGCTTCATGACTC -3 '
・ Primer 2R: 5'- GTCAACTTGCCATAAACCAGAG -3 '
・ Primer 3R: 5'- CCATAGCACACGCATACCAT -3 '
Amplified fragment with primers 1F and 2R: about 0.2 kb for wild type, about 0.3 kb when loxP sequence is included
Amplified fragment using primers 1F and 3R: about 3.5 kb in the wild type, about 5.4 kb when the loxP sequence and neo gene are included, and about 1.7 kb when exon 2 is deleted.

The result is shown in FIG. 2A. In FIG. 2A, (i) shows the results of ES cells. Each lane from left, wild-type cells before Cre expression ^{(ES Drp1 + / +),} Drp1 lox / lox cells before Cre expression, Drp1 ^{lox / lox} cells that expressed control gene (LacZ), after Cre expression Drp1 ^{lox / +} cells, Drp1 ^{lox / lox} cells after Cre expression. In ES cells, it was confirmed that the Drp1 gene with the loxP sequence inserted on both sides could be deleted by the expression of Cre protein. (ii) shows the result of MEF cells. Each lane is Drp1 ^{lox / lox} cells before Cre expression and Drp1 ^{− / −} cells obtained by Cre expression in order from the left. In Drp1 ^{− / −} MEF cells, it was confirmed that the Drp1 gene having loxP sequences inserted on both sides could be deleted. (iii) shows the result of the tail of Drp1-deficient mice. Each lane is Drp1 ^{+ / +} , Drp1 ^+/− , Drp1 ^{− / −} mouse in order from the left. (iv) shows the brain results of Drp1 ^{lox / lox} ; nestin-Cre mice. Each lane is a Drp1 ^{lox / +} mouse, a Drp1 ^{lox / lox} ; nestin-Cre mouse # 1, and a Drp1 ^{lox / lox} ; nestin-Cre mouse # 2 (confirming two mice of the same genotype) from the left.

Furthermore, expression of Drp1 protein was confirmed by Western blotting for protein extracts obtained from Drp1 knockout mice and knockout cells by a conventional method (FIG. 2B). As a control, the expression of Tom40, Fis1, and PDI proteins was also examined. In FIG. 2B, (i) shows the results of ES cells. Each lane is, from left to right, wild type cells before Cre expression (Drp1 ^{+ / +} ES), Drp1 ^{lox / lox} cells before Cre expression, wild type cells after Cre expression (Cre Drp1 ^{+ / +} ES), Cre expression Drp1 ^{lox / +} cells after, and Drp1 ^{lox / lox} cells after expression of Cre. (ii) shows the result of MEF cells. Each lane is Drp1 ^{lox / lox} cells before Cre expression and Drp1 ^{− / −} cells obtained by Cre expression in order from the left. (iv) shows the brain results of Drp1 ^{lox / lox} ; nestin-Cre mice. Each lane is a Drp1 ^{lox / +} mouse, a Drp1 ^{lox / lox} ; nestin-Cre mouse # 1, and a Drp1 ^{lox / lox} ; nestin-Cre mouse # 2 (confirming two mice of the same genotype) from the left. It was confirmed that the Drp1 gene product was definitely deleted in Drp1 knockout mice and knockout cells. In FIG. 2B, αDrp1, αTom40, αFis1, and αPDI are detection antibodies for Drp1, Tom40, Fis1, and PDI, respectively.

[Example 2] Analysis of Drp1-deficient cells For the Drp1 ^{lox / lox} and Drp1 ^-/- ES cells and MEF cells prepared according to Example 1, the intracellular mitochondrial morphology and distribution were observed. Observation of mitochondria was performed using a fluorescence microscope and an electron microscope, respectively. Fluorescence microscopy has been reported in each cell for mitochondria (mit), mitochondrial localized red fluorescent protein (su9-RFP: Eura et al., J Biochem (Tokyo). 2003 Sep; 134 (3): 333-344 The peroxisome (per) was stained with peroxisome-localized green fluorescent protein (GFP-SKL: a protein in which the peroxisome localization signal Ser-Lys-Leu was fused to the C-terminus of GFP). (FIGS. 3A-C). Each cell was further observed with an electron microscope by a conventional method (FIG. 3D). As a result of observation, as shown also in FIG. 3, in Drp1 ^{− / −} ES cells and Drp1 ^{− / −} MEF cells, mitochondrial division is suppressed and mitochondria are lengthened, and mitochondria are located in the periphery of the nucleus in the cell. It became clear that it was easy to accumulate. Furthermore, when the foreign Drp1 gene was introduced and expressed in Drp1 ^{− / −} ES cells, the morphology and localization of mitochondria approached that of Drp1 ^{lox / lox} cells, indicating that they were complemented (FIG. 3B). .

FIG. 4A shows a tubular form (tubular; black bar), a fragmented form (fragment; white bar), and an intermediate form (intermediate form) in observation of Drp1 ^{lox / lox} and Drp1 ^{− / −} ES cells. Intermediate; gray bars), the percentage of cells in which mitochondria were observed. In the cells with the loxP sequence inserted but retaining the Drp1 gene, many fragmented forms of mitochondria were observed, whereas in the homozygous knockout cells of the Drp1 gene, tubular forms of mitochondria were increased. On the other hand, in FIG. 4B, in the observation of Drp1 ^{lox / lox} and Drp1 ^{− / −} MEF cells, a highly connected form (highly connected; black bar), a tubular form (tubular; gray bar) And the percentage of the number of cells in which mitochondria in a fragmented form (fragment; white bar) were observed. Tubular forms of mitochondria were observed in cells that had the loxP sequence inserted but retained the Drp1 gene, whereas highly linked forms of mitochondria increased in homologous knockout cells of the Drp1 gene. Thus, in the homozygous knockout cells of the Drp1 gene, a larger volume of mitochondria was observed.

Furthermore, these cells were examined for cell proliferation (FIG. 5), mitochondrial membrane potential (FIG. 6A) and relative mitochondrial DNA (mtDNA) content (FIG. 6B). Cell proliferation was measured by cell count. Mitochondrial membrane potential was measured by quantification of mitochondrial membrane potential dependent fluorescent dyes. Total DNA was extracted from each cell by a conventional method, and the DNA samples obtained were serially diluted and used as a template to PCR-amplify the COXI gene (mtDNA) and Drp1 gene (genomic DNA), and then electrophorese them. The fractionated DNA was visualized, and the amount of each amplification was compared between the Drp1 ^{lox / lox} and Drp1 ^{− / −} samples, thereby relatively comparing the changes in mtDNA content and genomic DNA content. As a result, it is clear in the above points between cells that have loxP sequence inserted but retain normal Drp1 gene (Drp1 ^{lox / lox} ) and homozygous knockout cells of Drp1 gene (Drp1 ^-/- ) There was no significant difference. Traditionally, mitochondria were thought to proliferate with mitochondrial fission, so it was thought that knockout of Drp1 could inhibit mitochondrial growth and function. However, the above results differed from the expectation, indicating that the function of Drp1 is not essential for cell proliferation and mitochondrial proliferation, and maintenance of mitochondrial functionality.

[Example 3] Analysis of Drp1 whole body-deficient mouse fetus The Drp1 ^-/- mouse prepared in Example 1 is a Drp1 whole body deficient mouse in which the Drp1 gene is homozygous and disrupted in whole body cells. The development of Drp1 ^{− / −} mice was observed, the fetuses were removed from the pregnant mice and observed under a stereomicroscope, and the fetal paraffin-embedded sections were observed under the microscope.

As a result, Drp1 ^{− / −} mice became embryonic lethal before embryonic day 12.5 (E12.5) and were absorbed by the parent mouse body after embryonic day 15.5 (E15.5). In Drp1 ^{− / −} mice, developmental delay was observed from embryonic day 9.5 (E9.5) to embryonic day 11.5 (E11.5) (-/-in the photographs of FIGS. 7A to 7D). In Drp1 ^{− / −} mouse fetuses, formation of tissues such as brain, heart and liver was observed (FIG. 7E). As shown in FIG. 7E, cell growth was not stopped in E11.5 Drp1 ^{− / −} mouse embryos. Furthermore, in E11.5 Drp1 ^{− / −} mouse fetuses, increased apoptosis in neurons was observed (FIG. 7F). Thus, it was shown that the function of Drp1 gene is essential for the development of the mouse embryonic stage.

[Example 4] Analysis of nerve cell-specific Drp1-deficient mice According to Example 1, the development of knockout mice (nerve cell-specific Drp1-deficient mice) in which the Drp1 gene was specifically disrupted was observed. The fetus was observed in the same manner as in Example 3. As a control mouse, Drp1 ^{lox / +} ; nestin-Cre was used.

Nerve cell-specific Drp1-deficient mice (Drp1 ^{lox / lox} ; nestin-Cre) died immediately after birth (P0) without breastfeeding (FIG. 8A, lox / lox; nestin-Cre). In embryonic day 18.5 (E18.5) of neuron-specific Drp1-deficient mouse fetuses, brain sections were observed after paraffin embedding, enlargement of the ventricle and subarachnoid area due to brain regression, loss of subcortical white matter Was observed (FIGS. 8B and 8C).

  For E18.5 neuron-specific Drp1-deficient mouse embryos, apoptotic cells were detected by TUNEL staining using paraffin sections of the brain. Many TUNEL-positive cells were observed centering on the subcortical white matter region, indicating enhanced apoptosis (FIG. 8D).

  Furthermore, a large mitochondrial morphological change was observed in the brain of E18.5 neuron-specific Drp1-deficient mice. That is, it was recognized that the enlarged mitochondria were localized in a part of the cytoplasm in the brain (FIGS. 9A and B). This indicates that the Drp1 gene product induces mitochondrial division and contributes to the normal distribution of mitochondria in neurons, as well as the functional expression and viability of neurons. Unlike the results of Example 2, increased apoptosis was observed in mouse ES cells due to Drp1 deficiency, indicating that the Drp1 gene suppresses apoptosis (cell death) of neurons.

[Example 5] Drp1-deficient cell-derived neuronal dyssynaptic formation
The occurrence of synapse dysfunction in Drp1-deficient cell-derived neurons was confirmed as follows through induction of neuronal differentiation from Drp1 ^{− / −} ES cells.

First, Drp 1 ^{− / −} ES cells were cultured on a suspension culture dish for 2 days using a medium of LIF (−) to prepare an embryoid body (EB). The embryoid bodies were then cultured for 6 days in a culture medium containing insulin, transferrin, selenium chloride and human plasma fibronectin in DMEM / F12 medium. Then, the cell mass is peeled off with trypsin (TrypLE Select, GIBCO; derived from microbial fermentation, non-animal substitute product), and the culture dish coated with poly-L- (ornithine) / human fibronectin is used in DMEM / F12 medium with N2 The cells were cultured for 6 days in a medium containing supplement, B27 supplement, and human basic FGF. By such an operation, it is possible to induce differentiation of nerve cells having axons in the case of wild type ES cells. Therefore, the nerve cells were treated with anti-phosphorylated neurofilament antibody (SMI34: abcam) and β-tubulin III antibody (sigma), respectively, and mitofillin antibody, a mitochondria-specific antibody, was used. Was used to detect and observe mitochondria. As a control, similar to the wild type Drp1 ^{lox / lox} ES cells that have a homozygous normal Drp1 gene but have loxP sequences inserted on both sides of the Drp1 gene, the same neuronal differentiation induction experiment and antibody detection Went.

As a result, it was observed that neurons were induced from Drp1 ^{lox / lox} ES cells to form a central nervous system-like network (FIG. 10A, Drp1 lox / lox). On the other hand, in the case of Drp1 ^{− / −} ES cells, induced neurons were detected, but central nervous system-like network formation via synapses was not observed (FIG. 10A, Drp1 − / −). In addition, the number of β-tubulin III-positive cells detected per culture dish was significantly less in Drp1 ^-/- ES cell-derived cells than in Drp1 ^{lox / lox} ES cell-derived cells (Fig. 10B).

  From the above observations, it was shown that in Drp1-deficient neurons, synapse formation dysfunction occurred and a central nervous-like network could not be formed. In nerve cell-specific Drp1-deficient mice, the neuronal network is cut off due to the insufficiency of neuronal synapse formation, and neuronal apoptosis is enhanced, resulting in various neurodegenerative conditions. Therefore, it was shown that the nerve cell-specific Drp1-deficient mouse of the present invention can be used as a neurodegenerative disease model mouse having a phenotype that closely resembles the general pathology exhibited by neurodegenerative diseases.

[Example 6] Analysis of pancreatic β cell-specific Drp1-deficient mice According to Example 1, the generation of knockout mice (pancreatic β-cell-specific Drp1-deficient mice) in which the Drp1 gene was specifically disrupted was observed. As a control mouse, Drp1 ^{lox / +} ; RIP-Cre was used.

  Pancreatic β-cell-specific Drp1-deficient mice were able to grow smoothly after birth. The pancreas of a 6-week-old pancreatic β cell-specific Drp1-deficient mouse was isolated and histologically examined after preparing a paraffin section. Histological examination by HE staining showed no obvious morphological abnormalities in the pancreatic islets of Langerhans, and both pancreatic β cells and pancreatic α cells were found normally by immunostaining with anti-insulin antibody and anti-glucagon antibody.

Next, the paraffin section was subjected to anti-mitochondrial antibody staining (red) and nuclear staining with DAPI (blue) to confirm the intracellular localization of mitochondria. As shown in FIG. 11A, in the control mouse (Drp1 ^{lox / lox} mouse), mitochondria were found uniformly in almost all pancreatic β cells, whereas in the pancreatic β cell-specific Drp1-deficient mice, staining property in pancreatic β cells was observed. Was heterogeneous, ie, the mitochondrial subcellular localization was significantly altered. FIG. 11B shows an enlarged photograph of the photograph of FIG. 11A. The pancreatic β cells of the control mouse (wild type phenotype) stained the cytoplasm almost uniformly, whereas the pancreas of the pancreatic β cell-specific Drp1-deficient mice. In β-cells, many cells were localized in the cytoplasm, and no mitochondrial staining was seen on the same section. Thus, in the pancreas of pancreatic β cell-specific Drp1-deficient mice, pancreatic Langerhans islet cells were normally constructed, but it was observed that a large change occurred in the distribution of mitochondria in pancreatic β cells.

Furthermore, a glucose tolerance test was performed as follows using pancreatic β cell-specific Drp1-deficient mice (Drp1 ^{lox / lox} ; RIP-Cre mice) and control mice (Drp1 ^{lox / lox} mice). As a result of this glucose tolerance test, remarkable glucose tolerance was observed in pancreatic β-cell specific Drp1-deficient mice.

  First, each mouse (8 weeks old) was fasted for 16 hours, blood was collected from its tail vein, and blood glucose level and serum insulin level were measured in advance for the tail vein blood. Subsequently, 2 g / kg body weight of glucose was intraperitoneally administered to the mice. Blood was collected from the tail vein at 15 minutes and 30 minutes after glucose administration, and blood glucose level and insulin level were measured. Furthermore, blood was collected from the tail vein 60 minutes, 90 minutes, and 120 minutes after glucose administration, and the blood glucose level was measured.

  An example of the result is shown in FIG. 12A. As shown in FIG. 12A, in the control mouse, there is no significant difference between the 15-minute value and the 30-minute value, and a peak value is usually observed around 30 minutes, whereas in the pancreatic β cell-specific Drp1-deficient mouse, after 30 minutes. Thereafter, the blood glucose level increased rapidly and was still maintained at a high level even after 120 minutes (FIG. 12A, lox / lox RIP). FIG. 12B shows the blood insulin level at that time. In the pancreatic β cell-specific Drp1-deficient mice, the insulin level was not rapidly increased 15 minutes after glucose loading as observed in the control mice. There was a decrease in early insulin secretion compared to mice. The decrease in hyperglycemia and early insulin secretion that specifically occurs after glucose loading as shown in FIG. 12A was consistent with the symptoms of glucose-responsive insulin secretion deficiency typical of type 2 diabetes. In addition, as shown in the 0 minute value of FIG. 12A, the fasting blood glucose was not significantly different between the Drp-deficient mouse and the control mouse.

  For pancreatic β cell-specific Drp1-deficient mice and control mice, body weight was also measured in parallel with the above test. Pancreatic β-cell specific Drp1-deficient mice already showed impaired glucose tolerance at 8 weeks of age, but no significant difference in body weight was observed at 8 weeks of age compared to control mice (FIG. 13A).

  Moreover, insulin synthesis was confirmed in pancreatic β cells of pancreatic β cell-specific Drp1-deficient mice. Specifically, when a paraffin-embedded section of the pancreas was stained with an insulin antibody, the pancreas of a pancreatic β cell-specific Drp1-deficient mouse also resembled the pancreas of a control mouse (wild-type phenotype). Was observed. Furthermore, pancreatic β-cell specific Drp1-deficient mouse pancreas sections were continuously observed, the area of pancreatic islets of Langerhans was measured, and no significant difference was found between them when compared with control mice. From the above, it was found that insulin synthesis itself was normally performed even in pancreatic β-cell specific Drp1-deficient mice. Furthermore, when the pancreatic paraffin-embedded sections of control mice and pancreatic β cell-specific Drp1-deficient mice (KO mice) were double-stained with insulin antibody using glucagon antibody, the glucagon-positive α cells became β cells. It was found that the island is located on the edge so as to surround it, and from this the construction of La Isle Island can be seen normally (FIG. 13B).

  In this way, in pancreatic β cell-specific Drp1-deficient mice, the pancreatic β cells themselves were normally formed to form the pancreatic islets of Langerhans, and insulin was synthesized in the pancreatic β cells. Abnormal glucose tolerance was observed. Thus, it was shown that the Drp1 gene is involved in insulin secretion function through regulation of mitochondrial morphogenesis in pancreatic β cells, especially mitochondrial division.

  The knockout non-human mammal in which the function of the Drp1 gene according to the present invention is tissue-specifically deficient is a variety of disease states associated with mitochondrial dysfunction such as glucose-responsive insulin secretion failure, synapse formation failure, or It can be advantageously used as a model animal of the accompanying disease. The drug screening method using the knockout non-human mammal or a cell in which the function of the Drp1 gene derived therefrom is tissue-specifically deleted has not been previously known to be associated with mitochondrial dysfunction, for example, 2 It can be used to search for novel therapeutic agents that act by a mechanism completely different from that of conventional diabetes mellitus and neurodegenerative diseases.

FIG. 1 is a diagram showing a method for producing a Drp1 knockout mouse. A shows the Drp1 gene modification on the genome in the production of a conditional knockout mouse. B shows the flow of production of a knockout mouse or its cells deficient in Drp1 in a tissue-specific manner. FIG. 2 is a photograph showing the result of confirming the Drp1 gene modification in the prepared knockout mouse by PCR amplification (FIG. 2A) or Western blotting (FIG. 2B). (I) to (iv) in FIG. 2 correspond to (i) to (iv) in FIG. 1B. FIG. 3 is a photograph showing observation results of mitochondrial morphology in Drp1-deficient cells. A is a fluorescence micrograph of Drp1 ^{lox / lox} and Drp1 ^{− / −} ES cells. B is a photograph showing a cell in which Drp1 is exogenously expressed in Drp1 ^{− / −} ES cells. Compared with A, it can be seen that the cells are complemented. C is Drp1 ^{lox / lox} and Drp1 ^{- / -} fluorescence micrographs, D of the MEF cells Drp1 ^{lox / lox} and Drp1 ^{- / -} is an electron micrograph of the MEF cells. FIG. 4 is a graph showing the ratio of cells having mitochondria of each form for the Drp1 ^{lox / lox} and Drp1 ^{− / −} ES cells (A) and MEF cells (B). FIG. 5 is a graph showing the results of cell proliferation of Drp1 ^{lox / lox} and Drp1 ^{− / −} in ES cells (A) and MEF cells (B). FIG. 6A is a graph showing measurement results of mitochondrial membrane potential in Drp1 ^{lox / lox} and Drp1 ^{− / −} MEF cells. FIG. 6B is a photograph showing a relative comparison of mitochondrial DNA content in Drp1 ^{lox / lox} and Drp1 ^{− / −} ES cells. FIG. 7 is a photograph showing the developmental state of a Drp1 whole body deficient mouse fetus and its respective tissues. FIG. 8 is a photograph showing the active state of nerve cell-specific Drp1-deficient mice and their brain tissue. FIG. 9 is a photograph showing mitochondria in brain tissue in nerve cell-specific Drp1-deficient mice. A is a fluorescence micrograph and B is an electron micrograph. FIG. 10 is a photograph showing a state of synapse formation failure in the brain of a nerve cell-specific Drp1-deficient mouse. FIG. 11 is a photograph showing mitochondria in pancreatic β cells in pancreatic β cell-specific Drp1-deficient mice. FIG. 12 is a diagram showing blood glucose levels (A) and blood insulin levels (B) in pancreatic β cell-specific Drp1-deficient mice and control mice. FIG. 13 is a view showing a change in body weight of pancreatic β cells of pancreatic β cell-specific Drp1-deficient mice (A) and a photograph of a section of pancreatic islets of Langerhans (B). In FIG. 13A, black bars are homozygous pancreatic β cell-specific Drp1-deficient mice, gray bars are heterozygous pancreatic β-cell-specific Drp1-deficient mice, and white bars are control mice (Drp1 ^{lox / lox} mice). is there. In each photograph of FIG. 13B, the right is a pancreatic β cell-specific Drp1-deficient mouse (Drp1 ^{lox / lox} ; RIP-Cre mouse), and the left is a control mouse (Drp1 ^{lox / lox} mouse). Upper and lower rows are 5-week-old and 24-week-old mice, respectively. In FIG. 13B, red indicates immunohistochemical staining for insulin, and green indicates immunohistochemical staining for glucagon.

  SEQ ID NO: 1 is a loxP sequence.

  Sequence number 2-4 is a primer.

Claims (8)

A knockout non-human mammal , which is a glucose-responsive insulin secretion-deficient model animal, in which the function of Drp1 gene is specifically deficient in pancreatic β cells .   The knockout non-human mammal according to claim 1, wherein the function of the Drp1 gene is deficient by deletion, substitution and / or insertion of at least a part of the nucleotide sequence on the Drp1 gene or its expression control region.   The non-human mammal according to claim 1 or 2, which is a rodent. A synaptogenesis failure model animal, Drp1 gene function of you are deficient in neuronal cell-specific Roh Kkuauto non-human mammal. The knockout non-human mammal according to claim 4, wherein the function of the Drp1 gene is deficient by deletion, substitution and / or insertion of at least a part of the base sequence on the Drp1 gene or its expression control region. The non-human mammal according to claim 4 or 5, which is a rodent. A glucose-responsive insulin, characterized in that a test substance is administered to the knockout non-human mammal according to any one of claims 1 to 3 or pancreatic β cells deficient in the function of the Drp1 gene obtained therefrom. Screening method for secretion improving agent. An agent for promoting synapse formation, comprising administering a test substance to a knockout non-human mammal according to any one of claims 4 to 6 or a nerve cell deficient in the function of the Drp1 gene obtained therefrom. Screening method.

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