JP2007236347A - Central breathing disorder model/non-human animal - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a non-human animal that newly proves relation between deletion of DSCAM gene and crisis of central breathing disorder and elucidates crisis mechanism of central breathing disorder and makes a medicine associated with central breathing disorder, to provide a method for preparing a non-human animal, a method for screening a therapeutic agent for diseases caused by central breathing disorder, an assay method for diseases caused by central breathing disorder and an assay kit for diseases caused by central breathing disorder. <P>SOLUTION: The non-human animal exhibiting central breathing disorder has a genotype of deletion in Down syndrome cell adhesion molecule (DSCAM) gene. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  Central respiratory disorder is defined as a condition in which the central nervous system that controls respiratory movements in the brainstem and / or medulla does not operate normally and respiratory movements are abnormal. More specifically, examples of central respiratory disorder include congenital central hypoventilation syndrome (Ondine curse disease) and primary alveolar hypoventilaon syndrome (PAHS), which is an intractable disease. Central respiratory disorder is also suspected as a cause of sudden infant death syndrome and sleep apnea syndrome.

  However, the causative gene of central respiratory disorder has not yet been identified, and the onset mechanism of central respiratory disorder has not been elucidated. If the onset mechanism of central respiratory disorder is clarified, it is possible to expect effective treatment of the above-mentioned various syndromes and creation of drugs.

  By the way, cell adhesion molecules (CAMs) of the nervous system immunoglobulin superfamily are involved in various cell-cell interactions that are important for the development and function of the nervous system (including migration, neurite recognition, axon elongation, and axon repulsion). It is known to mediate. Among them, Down's syndrome cell adhesion molecule (DSCAM) is the largest member of the nervous system Ig superfamily isolated from human chromosome 21q22 (Non-patent Document 1). Non-Patent Document 1 and Non-Patent Document 2 identify mouse Dscam, which is widely expressed in the nervous system from fetal to adulthood.

  However, there is no knowledge showing the relationship between DSCAM and central respiratory disorder, and the DSCAM gene is not even listed as a candidate gene for central respiratory disorder.

Yamakawa et al. “DSCAM: a novel member of the immunoglobulin superfamily maps in a Down syndrome region and is involved in the development of the nervous system.” Hum. Mol. Genet. 7, 227-237 (1998) Agarwala et al. “Dscam is associated with axonal and dendritic features of neuronal cells.” J. Neurosci. Res. 66, 337-346 (2001)

  Therefore, in view of the above situation, the present invention relates to the mechanism of central respiratory disorder and central respiratory disorder by newly demonstrating the relationship between DSCAM gene deficiency and the development of central respiratory disorder. Useful non-human animals that enable drug discovery, methods for producing non-human animals, screening methods for therapeutic agents for diseases caused by respiratory central disorders, methods for examining diseases caused by respiratory central disorders, and respiratory central disorders An object is to provide a test kit for a disease.

The present invention that has achieved the above-described object includes the following.
(1) A non-human animal having a genotype deficient in a Down syndrome cell adhesion molecule (DSCAM) gene and exhibiting central respiratory disorder.
(2) The non-human animal according to (1), wherein the DSCAM gene is homo-deficient.
(3) The non-human animal according to (1), wherein the DSCAM gene is heterozygous.
(4) A method for producing a non-human animal having a genotype deficient in a Down syndrome cell adhesion molecule (DSCAM) gene and exhibiting central respiratory disorder, wherein the gene is used in a non-human animal embryonic stem cell Is selected or embryonic stem cells that have been rendered non-functional, a chimeric embryo is produced from the embryonic stem cells, the chimeric embryo is transplanted into the uterus of a temporary parent non-human animal, and a non-human pup is obtained by birth And obtaining a non-human animal having a genotype deficient in the DSCAM gene by selecting a chimeric non-human animal in which the gene is disrupted and mating the female and male of the chimeric non-human animal.
(5) The method according to (4), wherein a non-human animal in which the DSCAM gene is homo-deficient is obtained by the mating.
(6) The method according to (4), wherein a non-human animal in which the DSCAM gene is hetero-deficient is obtained by the mating.
(7) A step of administering a candidate substance to the non-human animal according to any one of (1) to (3), a step of measuring at least one data of (1) to (4) above,
(1) Data showing respiratory rhythm of non-human animals (2) Data showing C4 anterior root activity (3) Data showing anterior respiratory neuron activity (4) Data showing neurite outgrowth of dorsal root ganglion neuron culture When any one or more of the following (1) to (4) is significantly improved as compared with the data measured using the non-human animal not administered with the candidate substance, the candidate substance is treated as respiratory center disorder And a method for screening a therapeutic agent for a disease caused by respiratory center disorder, comprising the step of identifying the therapeutic agent for a disease caused by the disease.
(8) The screening method according to (7), wherein the disease is congenital central hypoventilation syndrome.
(9) The screening method according to (7), wherein the disease is accompanied by Hirschspring disease.
(10) A disease caused by respiratory central disorder, including a step of collecting a biological sample from a subject, and a step of detecting a mutation of Down syndrome cell adhesion molecule (DSCAM) gene in the collected biological sample. Inspection method.
(11) A diagnostic kit for a disease caused by respiratory center disorder, comprising a reagent for detecting a mutation in a Down syndrome cell adhesion molecule (DSCAM) gene in a biological sample collected from a subject.

  Hereinafter, the present invention will be described in more detail with reference to the drawings.

1. DSCAM gene In the present invention, the Down syndrome cell adhesion molecule (DSCAM) gene is a member of the nervous system immunoglobulin superfamily that is deeply involved in the development and differentiation of the nervous system.・ It is expressed in neural tissues that occur early, such as dorsal root ganglia. The DSCAM gene has been identified in mammals such as humans, mice, and rats, insects such as Drosophila, moss, silkworms, fish such as zebrafish, and the like. The DSCAM gene has been confirmed to be expressed in various organisms as described above, and can be easily isolated and identified in an unidentified organism according to a conventional method. However, various splicing variants are known for the DSCAM gene, and the encoded protein is not one kind.

  For example, as a human-derived DSCAM gene, a DSCAM gene encoding a protein having the amino acid sequence shown in SEQ ID NO: 1 can be mentioned. An example of a mouse-derived DSCAM gene is a protein having the amino acid sequence shown in SEQ ID NO: 2.

2. DSCAM gene knockout / non-human animal In the present invention, a non-human animal exhibiting central respiratory disorder can be produced by deleting the DSCAM gene described in the above "1". Here, the non-human animal means an animal excluding humans, for example, mammals excluding humans (mouse, rat, rabbit, dog, cat, monkey, sheep, pig, cow, horse, etc.), birds (for example, Chicken, etc.), amphibians (eg, frogs), reptiles or insects.

  A technique for producing a non-human animal is not particularly limited, and a conventionally known technique can be appropriately used. Conventionally known methods include, for example, “the latest technology of gene targeting” (Yoji Ken Yodosha), “gene targeting” (translated by Tetsuo Noda, Medical Science International), Science 244: 1288-1292, 1989, etc. It is disclosed.

For example, a chimeric non-human animal is obtained by injecting embryonic stem cells (hereinafter also referred to as ES cells) in which at least one allele DSCAM gene is disrupted by homologous recombination into a blastocyst of a fertilized egg. The resulting chimeric animal can be crossed with the corresponding wild-type non-human animal to produce a heterozygous non-human animal (hereinafter also referred to as DSCAM ^+/- ) in which only one of the alleles is destroyed. it can. Furthermore, these obtained heterozygous non-human animals can be mated to produce homozygous non-human animals (hereinafter also referred to as DSCAM ^{− / −} ) in which both alleles are disrupted. Wild type is expressed in DSCAM ^{+ / +} .

  As an ES cell, E14, CCE, J1, or T2 can be used in the case of a mouse. Homologous recombination of ES cells can be performed, for example, by introducing recombinant DNA into which any DNA sequence capable of interfering with the expression of the DSCAM gene is introduced into ES cells. As an arbitrary DNA sequence capable of interfering with the expression of the DSCAM gene, a drug resistance gene (for example, neomycin resistance gene (neo)) that can confirm introduction of recombinant DNA into ES cells is preferably used. A gene (β-galactosidase (lacZ)) or the like is preferably used, but is not particularly limited.

  Examples of the DNA introduction method include an electroporation method and a microinjection method. The disruption of the DSCAM gene in ES cells can be confirmed by, for example, PCR and Southern blot hybridization according to a conventional method.

  Examples of a method for producing a chimeric non-human animal having ES cells in which the DSCAM gene is disrupted by homologous recombination include a blastocyst microinjection method. The blastocyst injection method involves microinjecting a DSCAM gene-disrupted ES cell into a non-human animal blastocyst and transplanting the treated blastocyst into a pseudopregnant non-human animal to obtain a desired chimeric non-human animal. Can be created.

  A heterozygous non-human animal obtained by mating the chimeric non-human animal obtained as described above with a corresponding wild-type non-human animal, or obtained by further mating the heterozygous non-human animal. The homozygous non-human animal obtained can be confirmed for its genotype by, for example, Southern blotting using DNA collected from the tail. In addition, the presence or absence of expression of the DSCAM gene can be confirmed by RNA blot analysis.

The produced knockout non-human animal is surprisingly found to develop central respiratory disorder, and can be used as a central respiratory disorder model animal. In particular, DSCAM ^-/- has the characteristics that it is born normally, but almost all individuals die from respiratory disorder within 24 hours. In particular, DSCAM ^+/− has the characteristics that it is born normally and grows similarly to DSCAM ^{+ / +} , but slightly develops respiratory disorder. As described in “1” above, it is known that the DSCAM gene is expressed in various neural tissues, but the characteristics of the above-described knockout non-human animals (DSCAM ^{− / −} and DSCAM ^+/− ). Is not predictable at all.

In addition, DSCAM ^{− / −} has a feature that the size of medulla and spinal cord is significantly enlarged compared to wild type DSCAM ^{+ / +} . Also, 50% of patients with congenital central hypoventilation syndrome (CCHS) have Hirschsprung disease (HSCR). HSCR is a congenital disease characterized by a loss of enteric ganglion along the variable-length intestine (Croaker et al. (1998) “Congenital central hypoventilation syndrome and Hirschsprung's disease” Arch. Dis. Child 78, 316-322. .). The pathology of HSCR is thought to be due to the failure of neural crest cells to migrate. The DSCAM gene is known to be expressed in the intestinal myenteric plexus derived from the neural crest (Yamakawa et al. (1998) “DSCAM: a novel member of the immunoglobulin superfamily maps in a Down syndrome region and is involved in the development of the nervous system ”Hum. Mol. Genet. 7, 227-237.). In addition, one of the change factor regions of HSCR has been reported on chromosome 21q22 (Puffenberger et al. (1994) “Identity-by-descent and association mapping of a recessive gene for Hirschsprung disease on human chromosome 13q22” Hum. Mol. Genet. 3, 1217-1225). This DSCAM gene is located within the region reported in the paper. Therefore, it is suggested that the DSCAM gene is also involved in the development of HSCR.

3. Use of DSCAM gene knockout / non-human animal The DSCAM gene knockout / non-human animal described in “2” above should be used as a model animal for central respiratory disorder by utilizing the characteristics of developing central respiratory disorder. Can do. Specifically, a DSCAM gene knockout / non-human animal can be used in a screening method for identifying a therapeutic agent for a disease caused by respiratory central disorder.

Specifically, first, a candidate substance is administered to a DSCAM gene knockout / non-human animal. Here, the candidate substance means a substance that can be a candidate for a therapeutic drug for central respiratory disorder. The candidate substance may be a known substance or an unknown substance. The candidate substance may be any substance such as a protein, peptide, nucleic acid molecule, pseudo-nucleic acid molecule, low molecular compound, high molecular compound, organic compound, and inorganic compound. In particular, DSCAM ^-/- died 24 hours after birth, so it should be administered before death. In contrast, since DSCAM ^+/- grows in the same manner as the wild type, it can be administered at various stages of growth.

  Next, in this screening method, the effect of improving a central respiratory disorder in a non-human animal by DSCAM gene knockout by administration of a candidate substance is confirmed. For example, DSCAM gene knockout after administration, respiratory rhythm in non-human animals, or respiratory center function is monitored. More specifically, at least one data of the following (1) to (4) is measured.

(1) DSCAM gene knockout / data showing respiratory rhythm of non-human animals (2) data showing C4 anterior root activity (3) data showing prerespiratory (Pre-I) neuron activity (4) dorsal root ganglion neurons Data showing neurite outgrowth in culture

  The method for measuring the respiratory rhythm shown in the above (1) is not particularly limited, and examples thereof include a method using whole body plethysmography (Kuwaki et al. (1996) “Impaired ventilatory responses to hypoxia and hypercapnea in mutant mice deficient). in endothelin-1 ”Am. J. Physiol. 270, 1279-1286). In addition, the method for measuring the C4 anterior root activity shown in (2) above is not particularly limited, but using a medulla and spinal cord for in vitro preparation, respiratory-like activity corresponding to the inspiration rhythm is exhibited by a glass capillary suction electrode and The measurement can be performed through a high-pass filter with a time constant of 0.3 seconds (Shirasawa et al. (2000) “Rnx deficiency results in congenital central hypoventilation” Nat. Genet. 24, 287-290, Suzue et al. (1984) “ Respiratory rhythm generation in the in vitro brain stem-spinal cord preparation of the neonatal rat ”J. Physiol.354, 173-183 and Onimaru et al. (1988)“ Primary respiratory rhythm generator in the medulla of brainstem-spinal cord preparation from newborn rat "See Brain Res. 445, 314-324). The method for measuring Pre-I neuron activity shown in (3) above is not particularly limited, but the average membrane potential trajectory is measured by applying an optical recording system using a brainstem-spinal cord preparation stained with a voltage-sensitive dye. (See Onimaru et al. (2003) “A novel functional neuron group for respiratory rhythm generation in the ventral medulla” J. Neurosci. 23, 1478-1486). In addition, Pre-I neuron activity can be measured by, for example, measuring the membrane potential of inspiratory neurons in the ventral lateral medulla using the whole cell patch clamp method (Onimaru et al. (1992) “Whole cell recordings from respiratory neurons in the medulla of brainstem-spinal cord preparations”. Using isolated from newborn rats ”Pflugers. Arch. 420, 399-406 and Arata et al. (1990)“ Respiration-related neurons in the ventral medulla of newborn rats in vitro ”Brain Res. Bull. 24, 599-604). Can be measured. The method for measuring neurite outgrowth of the dorsal root ganglion neuron culture shown in (4) above is not particularly limited, but the dorsal root ganglion (DRG) is played on a petri dish coated with Dscam-Fc or L1cam-Fc. And imaging the culture (Kamiguchi et al. (2001) “The role of endocytic l1 trafficking in polarized adhesion and migration of nerve growth cones” J. Neurosci. 21, 9194-9203).

  Next, in the present screening method, any one or more of the data (1) to (4) is compared with data measured using the non-human animal not administered with the candidate substance. As a result of comparison, when a candidate substance is administered, if any of the data is significantly improved, the candidate substance is identified as a therapeutic agent for a disease caused by respiratory center disorder.

4). Utilization of the DSCAM gene As described above, it has been demonstrated that a deficiency in the DSCAM gene causes central respiratory disorder. Based on this finding, the DSCAM gene is deeply involved in the mechanism of central respiratory disorder. Therefore, the diagnosis of central respiratory disorder can be supported by examining the DSCAM gene in patients suspected of having central respiratory disorder. That is, based on the knowledge described above, in a biological sample collected from a subject, a respiratory center disorder including a method for detecting a respiratory center disorder including a step of detecting a DSCAM gene mutation and a reagent for detecting a DSCAM gene mutation A diagnostic kit can be provided.

  Here, the DSCAM gene mutation means not only a mutation such as a DSCAM gene deletion and a missense mutation, but also a change in the expression level of the DSCAM gene. That is, a mutation in the promoter region that controls the expression of the DSCAM gene and a transcriptional control factor of the DSCAM gene is also defined as a mutation in the DSCAM gene. By detecting a mutation in the DSCAM gene in this sense, it becomes possible to diagnose central respiratory disorder in a subject.

  Examples of the reagent for detecting a mutation in the DSCAM gene include a probe that specifically hybridizes to the human-derived DSCAM gene shown in SEQ ID NO: 1 and a primer set that specifically amplifies the DSCAM gene. An antibody against the DSCAM protein encoded by the DSCAM gene can also be used as a reagent for detecting a mutation in the DSCAM gene. These probes, primer sets and antibodies can be used for detecting deletion of the DSCAM gene and changes in its expression level.

  On the other hand, a missense mutation in the DSCAM gene is specific to a probe that specifically hybridizes to the mutant DSCAM gene having the mutation, a primer set that specifically amplifies the mutant DSCAM gene, and a mutant DSCAM protein encoded by the mutant DSCAM gene. Can be detected using specific antibodies.

Furthermore, as an example, mutation of the DSCAM gene can be used for a heteroduplex method combined with a DHPLC (denaturing HPLC) method (WAVE ^™ DNA Fragment Analysis System (Transgenomic, Inc., San Jose, CA)). The instrument includes an autosampler, a capillary column packed with a high-resolution micro-pericular matrix, software for predicting and inputting separation conditions and outputting separation results, and a display.

  In this method, for at least one exon out of 33 exons in the DSCAM gene, a pair of primers that specifically amplify the exon is designed. At this time, as a pair of primers, all 33 exons may be designed, or only a part of exons may be designed. In particular, in a patient who develops a central respiratory disorder, if it is clear that a specific region of the DSCAM gene has a mutation, a primer may be designed for an exon containing the mutation.

  In this method, a pair of primers designed in this manner is used to amplify an exon in the DSCAM gene, and a solution containing the amplified DNA fragment is heat-treated to form a heteroduplex. Then, by eluting the solution containing the heteroduplex on the principle of chromatogradation, the elution peak appearing at a different position is compared with the elution peak of the wild-type homoduplex containing no mutation. Identify as According to this method, it is possible to detect the presence or absence of a mutation in a specific exon of the DSCAM gene using patient-derived genomic DNA. Then, by detecting the presence or absence of the mutation, useful information for supporting diagnosis of central respiratory disorder for the patient can be acquired.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is limited to these Examples and is not interpreted.

[Example 1] Preparation of Dscam gene knockout mouse In this example, as shown in Fig. 1a, a Dscam gene knockout mouse from which the first exon of the Dscam gene (indicated by a black square in Fig. 1a) is deleted. Produced. The mouse PAC library (RPCI-21: mouse 129s6 / SvEvTAC; Children's Hospital Oakland Research Institute, Oakland, CA, USA) is screened with probes to obtain genomic clones that transform the first exon, and two independent types Clones (639K11 and 671I13) were identified. The genomic structure of the region around 30 kb containing the first exon was confirmed by Southern blot analysis using several restriction enzymes and probes in these two clones.

  A targeting construct using the Cre-loxP system was prepared as follows. First, the 2.8 kb EcoRI-SpeI fragment of the 639K11 clone was subcloned into the EcoRI-SpeI site of the plasmid vector pBluescript II SK (-) (Stratagene Inc., La Jolla, CA, USA). Subsequently, the blunt-ended 2.8 kb EcoRI-SpeI fragment excised by EcoRI and SpeI digestion was cloned into the blunt-ended SalI site of the targeting construct to create the 3 'arm of the construct. The 5 'arm was created by inserting the 8.8 kb blunt-ended BamHI-EcoRI fragment of the 639K11 clone into the blunt-ended HbaI site followed by the loxP fragment into the MluI site, thus targeting with neomycin phosphotransferase (neo) A construct was made.

  In FIG. 1a, Ac is AccI, B is BamHI, EI is EcoRI, EV is EcoRV, Hc is HincII, Hd is HindIII, M is MluI, and Sp is SpeI.

The targeting construct was then linearized with NotI and electroporated into approximately ⁷ × 10 ⁷ E14ES cells at 3 μF and 800 V using Gene-Pulser (BioRad Laboratories, Hercules, Calif., USA). Transfected cells were plated into neomycin-resistant, mitomycin-treated mouse embryonic fibroblasts (MEF) in a 10 cm dish. One day after plating, positive selection was performed in the presence of geneticin (G418; 125 μg / ml). Resistant clones were selected on day 9 and then grown in 24-well plates pre-plated with MEF. EcoRV digested genomic DNA from individual clones was analyzed by Southern blot using a 356 bp probe (shown as P2 in FIG. 1a) derived from the genomic sequence immediately upstream of the targeting construct (FIG. 1b). The probe was prepared by PCR. Positive clones were further confirmed by hybridizing SpeI digested genomic DNA to a 5-terminal probe (shown as P1 in FIG. 1a. 385 bp PCR amplified fragment).

  Approximately 15 ES cells from each of the two target clones (A38 and A151) were injected into C57BL / 6 blastocysts. An average of 15 injected blastocysts were transferred to pseudopregnant female recipient ICR mice. All ES clones produced a slight agouti-colored mouse in more than 50%. Two types (one for each clone) of male chimeras were mated with C57BL / 6 females to enable detection of germline transmission using Agouti's coat color marker. As measured by Southern blot analysis, 52% (17/33) of agouti offspring were positive for insertion. Heterozygous flox-neo female mice were crossed with CAG-cre males to produce heterozygous knockout mice.

  Experimental procedures and animal rearing conditions were approved by the RIKEN Institute's Animal Experiments Committee, and all animals were cared for and treated humanely according to institutional guidelines for animal experiments.

The resulting heterozygous knockout mice (Dscam ^+/− mice) were fertile and developed almost normally. In addition, when homozygotes were produced by crossing between heterozygotes, the genotypes of all offspring immediately after birth matched the Mendelian segregation ratio (wild type, 220/835, 26.3%; Dscam ^+/− 420/835, 50.3%; Dscam ^{− / −} , 195/835, 23.4%), indicating that the Dscam ^{− / −} mice were not embryonic lethal. However, most Dscam ^{− / −} mice died within 24 hours after birth and rarely survived for more than 48 hours.

[Example 2] Analysis of Dscam gene knockout mice
Northern blot analysis Northern blot analysis was performed to confirm the expression of the DSCAM gene in the resulting knockout mice. Brain tissue was cut from P0 mice. Total RNA was extracted using Trizol reagent (Invitrogen Corp., Carlsbad, Calif., USA), and mRNA was isolated using μMACS mRNA Isolation Kit (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). 3 μg of poly (A) RNA was filled in each lane of a 1% agarose formaldehyde gel, and RNA separated according to size was transferred to a nylon membrane (Biodyne; Pall Bio Support, East Hills, NY, USA). For hybridization, DNA probes were labeled with [α- ³² P] dCTP and hybridized overnight in ExpressHyb hybridization solution (Clontech, Palo Alto, CA, USA) as recommended for the product. Finally, the blot was washed in 0.1 × SSC / 0.1% SDS at 55 ° C. and exposed to X-ray film at −80 ° C.

  The results are shown in FIG. In FIG. 1c, Ex.1 means exon 1 part (1-496bp), EC means extracellular part (2299-2626bp), TM means transmembrane part (5210-5410bp), CP means cell part (5385-5637bp). In this example, wild type (indicated as + / + in FIG. 1c), Dscam +/− (indicated as +/− in FIG. 1c) and Dscam − / − mouse (indicated as − / − in FIG. 1c) are analyzed. Went.

As shown in FIG. 1c, Northern blot analysis revealed no Dscam transcripts in Dscam ^{− / −} offspring brains and decreased Dscam gene expression in Dscam ^+/− offspring brains. It was confirmed.

For reference to RNA in situ hybridization, expression of Dscam mRNA was confirmed by RNA in situ hybridization immediately after birth of a wild type mouse (Dscam ^{+ / +} mouse) (ie, when Dscam ^{− / −} mouse suddenly died). RNA in situ hybridization was performed on cryosections using a cRNA probe labeled with digoxigenin. First, the sections were washed with 0.1 M PB (phosphate buffer) and fixed with 4% PFA for 30 minutes. The sections were then washed with 0.1M PB and then permeabilized with 0.3% Triton X-100 in 0.1M PB for 5 minutes. These were washed with 0.1M PB for 5 minutes, followed by 10 minutes with 0.1N HCl in 1 × SSC. They were then acetylated with 0.25% acetic anhydride in 0.1M triethanolamine for 10 minutes and washed with 0.1M PB. The sections were then prehybridized in hybridization buffer at 55 ° C. in the absence of cRNA probe for 16 hours and hybridized with 10 μg / ml cRNA probe (50% formamide, 5 × SSC, 0.1% N-lauroylsarcosine, 0.02% blocking agent) and hybridized overnight at 55 ° C. Sections were washed 3 times at 55 ° C. in 2 × SSC containing 50% formamide for 1 hour each. Hybridization was detected using NBT / BCIP (Roche Diagnostics) using anti-DIG Fab (Roche Diagnostics) conjugated with alkaline phosphatase.

  The results are shown in FIGS. FIG. 2a is a photograph of a sagittal section of a P0 mouse brain hybridized with a Dscam antisense probe. FIG. 2b is an enlarged photograph of the region (corresponding to the medulla oblongata) surrounded by the solid line in FIG. 2a. FIG. 2c is a photograph of a coronal section having a solid line position in FIG. 2b as a cutting level. FIG. 2d is a schematic diagram of respiratory neuron distribution in the medulla oblongata. In FIG. 2c and FIG. 2d, the area surrounded by a solid line indicates the position of the respiratory center. In FIG. 2d, white circles or black circles indicate the recording sites of pre-inspiratory (Pre-I) or inspiratory (I) neurons, respectively.

  As shown in FIG. 2a, Dscam mRNA was widely expressed in the cerebral cortex, hippocampus, thalamus, pons, olfactory bulb, and was predominantly expressed in the respiratory center of the medulla oblongata. As indicated by the arrow in FIG. 2b, Dscam was expressed in respiratory rhythm-forming neurons in the ventral medulla. As shown in FIGS. 2c and 2d, Dscam mRNA was expressed at sites where Pre-I and I neurons were activated.

Brain Morphological Analysis and Immunohistochemistry To evaluate histological abnormalities in the resulting knockout mice, brain morphology isolated from P0 Dscam ^{− / −} mice and their wild-type littermates was investigated. Anesthetized Dscam ^{− / −} mice or wild type mice were transcardially perfused with 4% paraformaldehyde in 0.1M PB. 20 μm cryosections were stained with 0.9% toluidine blue (Nissl staining). In addition, 34 types of markers (SMI311, SMI52, SMI35, NF150, NFL, NF-MH, 2H3, 3A10, CD57, 5HT, Nestin, RAT401, OLIG2, NeuN, v-glut2, NK1R, GLAST, Brn3a, Peripherin, CR50 , GAD65, glutamate, GABA, calretinin, AChE, ChAT, Pax2, Pax6, GluR2, Phox2b, TH, μ-opioid R1, somatostatin and spot). For immunohistochemical analysis, antibodies were diluted with PBS / 0.01% Triton X-100 and 5% bovine serum albumin.

FIG. 3a shows a ventral view of the brains of Dscam ^{− / −} and wild type mice on day 0 after birth. It can be seen that the medulla and spinal cord of Dscam ^{− / −} mice are larger than those of wild type mice. FIG. 3b shows the results of measuring the width of the cerebrum (solid line marked with ^* in FIG. 3a) and the medulla oblongum (solid line marked with ^** in FIG. 3a). The measurement results were compared by t-test. In FIG. 3b, error bars indicate SEM. As shown in FIG. 3b, the widths of the cerebrum and medulla were 6.05 ± 0.07 mm and 3.11 ± 0.04 in the wild type (n = 7), respectively. In contrast, it was 6.20 ± 0.07 and 3.35 ± 0.03 mm in Dscam ^{− / −} mice (n = 7), respectively. P = 0.188 for the cerebrum and p = 0.0004 for the medulla oblongata. The medulla and spinal cord of Dscam ^{− / −} mice were 7.8% larger than those of wild-type littermates.

Figures 3c-j show the results of Nissl staining of coronal sections of medulla oblongata (Figures 3c, d, g and h) and spinal cord (Figures 3e, f, i and j). The medulla of Dscam ^{− / −} mice (FIGS. 3d and h) are both on the dorsoventral axis (FIGS. 3c and d) and the inner and outer axes (FIGS. 3g and h) than those of wild-type mice (FIGS. 3c and g) You can see that the size is large. In addition, the spinal cords of the Dscam ^{− / −} mice (FIGS. 3f and j) are those of the dorsal abdominal axis (FIGS. 3e and f) and the internal and external axes (FIGS. 3i and j) of the wild type mouse (FIGs. 3e and i). It can be seen that the size is large in both cases. These results show that there are no obvious structural defects in these tissues of Dscam ^{− / −} mice, and medullary and spinal cord cell sizes are similar between Dscam ^{− / −} and wild type cells, This suggests that the number increased with Dscam ^{− / −} .

Respiratory analysis The appearance of Dscam ^{− / −} mice produced in Example 1 appeared normal immediately after birth and their body weight was not statistically different from that of wild type and Dscam ^+/− littermates (data not shown) ). However, we observed that Dscam ^{− / −} offspring showed difficulty in seizures or breath-like movements. Also, there are few Dscam ^{− / −} offsprings with milk in the stomach, indicating that most Dscam ^{− / −} offspring are not breastfeeding. These phenotypes in Dscam ^{− / −} offspring and Dscam mRNA are predominantly expressed in the medullary respiratory center, so Dscam ^{− / −} and Dscam ^+/− offspring and wild type using whole body plethysmography Spontaneous respiratory activity in litters was investigated.

Specifically, whole body plethysmography was performed on P0 mice under the age of one day (see Kuwaki et al. (1996) above). Respiration frequency, tidal volume, and minute volume were calculated from 50 to 70 breath cycles during rest breathing. The result is shown in FIG. 4a. As shown in FIG. 4a, Dscam ^{− / −} birth pups have irregular and frequent apnea, whereas wild littermates have regular and constant respiratory rhythms, and Dscam ^+/− is also slightly abnormal. It became clear to show.

The medulla and spinal cord for in vitro preparations were also isolated from P0 mice that had been fully ether anesthetized as previously described (Shirasawa et al. (2000), Suzue et al. (1984) and Onimaru et al., Supra). (See 1988). Respiratory-like activity corresponding to the inspiration rhythm was monitored at the C4 / C5 anterior root via a glass capillary suction electrode and a high-pass filter with a time constant of 0.3 seconds. Values are shown as mean ± SD. Statistical significance was determined by analysis of variance (ANOVA) for all three genotypes. Significant differences between groups were identified using a post hoc Bonferroni test. Membrane potentials of inspiratory neurons in the ventral lateral medulla (above Arata et al. (1990)) were recorded using a conventional whole cell patch clamp method (above Onimaru et al. (1992)). The membrane potential was recorded with a single electrode membrane potential fixing amplifier (CEZ-3100, Nihon Kohden, Tokyo, Japan) after compensation of continuous resistance (25-50 mol / LΩ) and capacitance. The result is shown in FIG. 4b. As shown in FIG. 4b, low amplitude C4 activity indicating apnea was observed in Dscam ^{− / −} mice (arrow in FIG. 4b). From the results shown in FIG. 4b, it was shown that the abnormal breathing pattern of Dscam ^{− / −} mice (FIG. 4a) was caused by the respiratory rhythm formation center located in the ventral medulla.

Optical recording method To visualize the spatio-temporal pattern of respiratory neuronal activity in ventral medulla, we applied an optical recording system using a brainstem-spinal cord preparation stained with a voltage-sensitive dye (Onimaru et al. (2003), supra). ). First, the medulla-spinal cord preparation is prepared in a modified Krebs solution containing a voltage-sensitive fluorescent dye, Di-2-ANEPEQ (33 μg / ml, Molecular Probe, Eugene, OR) at a ratio of 95% O ₂ and 5% CO ₂ . And incubated at room temperature for 30 minutes. After staining, the preparation was placed on the ventral surface in a 2-mL perfusion chamber fitted with a fluorescence microscope (THT-75 microscope setup, Scimedia Ltd., Tokyo, Japan). Neuron activity is recorded with a 510-550nm excitation filter, dichroic mirror, and a tungsten-halogen lamp (150W) using an optical recording device (MiCAM02; Brain Vision Inc., Saitama, Japan) using a voltage-sensitive dye. Detection was through a 590 nm absorption filter. The CCD base camera consists of 192x128 pixels (33.3x37.5μm / pixel). Optical images were obtained every 20 milliseconds. The acquisition time of inspiratory activity was 5.12 seconds, consisting of 3.84 seconds before the start of C4 inhalation group and 1.28 seconds after the start. The acquired image was an average of 50 respiratory cycles. The optical activity of each type of neuron was measured as the mean ΔF / F value including 78 pixels. The change in fluorescence was expressed as a ratio (%, partial change) (ie, fluorescence intensity relative to the fluorescence intensity of the reference image). The differential image processed with the 2x2 pixel software-spatial filter was represented by a pseudo-color display where red corresponds to a decrease in fluorescence (which indicates membrane depolarization). BV_analysis (BrainVision Inc., Saitama, Japan) was used for data acquisition and analysis.

  The results are shown in FIGS. A ΔF / F pseudocolor image from the C4 inspiration cluster at -1500 ms (FIGS. 5a and b) or 0 ms (FIGS. 5c and d) was superimposed on the ventral surface of the medulla oblongata. The optical image result is the average of 50 respiratory cycles induced by C4 inspiratory activity. In FIG. 5, the red circle indicates the most prominent region of Pre-I (pre-inspiratory) neuron activity, and the blue circle indicates the most prominent region of I (inspiratory) neuron activity. FIGS. 5e and f quantify the optical response within the red and blue circles (78 pixels) as red (upper profile in FIGS. 5e and f) and blue traces (middle profile in FIGS. 5e and f), respectively. Indicated. In FIGS. 5e and f, the lower trace (black) indicates C4 anterior root inhalation activity. Each point in the image is indicated by a vertical dotted line in the graph.

As shown in FIGS. 5a and e, in wild-type mice, the mean membrane potential trajectory of pre-inspiratory (Pre-I) neuron activity is inhaled in a limited region of the rostral ventrolateral part (RVLM). Hundreds of milliseconds appeared at the facial nucleus level before the onset of sexual (Pre-I) nerve activity. Subsequently, as shown in FIGS. 5c and e, respiratory neuronal activity was observed to propagate to the center and tail in the ventral medulla oblongata. In contrast, as shown in FIGS. 5b and f, it was found that in Dscam ^{− / −} mice, the mean membrane potential trajectory of Pre-I neuronal activity in RVLM disappeared during the pre-inspiration phase. These results indicate that respiratory dysfunction in Dscam ^{− / −} mice results from abnormalities in Pre-I neurons.

  Furthermore, in order to investigate the basic properties of long-lasting activity of Pre-I neurons (upper profile in Fig. 5e), several of them in the corresponding region of RVLM from wild-type mice using the conventional whole-cell patch clamp method The membrane potential of neurons was recorded. As a result, as shown in FIG. 6, it was observed that a plurality of subtypes of membrane potential of Pre-I neuron activity appeared continuously before the start of I-neuron activity. These results suggest that Pre-I neurons consist of multiple subtypes, which are activated sequentially. They bind to each other, relay their activity, and then transfer activity to I-neurons.

Neurite outgrowth assay The homophilic binding-dependent promoting activity of Dscam in neurite outgrowth was also investigated.
The dorsal root ganglia (DRG) excised from P0 mice were treated with trypsin-EGTA (Life Technologies) at 37 ° C. for 30 minutes and dissociated by grinding with a p20 micropipette man. Dissociated cells were resuspended in RPMI1640 medium (Life Technologies) supplemented with 10% fetal calf serum (Life Technologies), 1% PSF (Life Technologies) and 20 ng / ml NGF (Promega), and Dscam-Fc or L1cam ( The cells were plated on a glass petri dish coated with the cell adhesion molecule L1) -Fc. Coat a petri dish coated with CAM-Fc in order with 0.1 mg / ml poly-D-lysine (70-150 kD; Sigma Aldrich), 40 μg / ml anti-Fc antibody (Sigma Aldrich) and Dscam-Fc or L1cam-Fc. Prepared by. The production of Dscam-Fc or L1cam-Fc was performed as described previously (Kamiguchi and Yoshihara (2001) above). Prior to fixation with 4% formaldehyde, cells were cultured in a humidified incubator for 7 hours at 37 ° C., 5% CO ₂ .

  Images of DRG neurons taken with a cooled digital CCD camera (CoolSNAP HQ; Photometrics) were analyzed using MetaMorph version 4.6 (Universal Imaging). The length of the neurite was measured as the distance between the tip of the longest neurite and the outer surface of the cell body where the neurite appeared.

  The results are shown in FIGS. 7a and b. In FIG. 7a, the number of samples of Dscam-Fc is n = 465 or n = 348, and the number of samples of L1cam-Fc is n = 327 or n = 335. Moreover, the negative control has shown the result at the time of using the petri dish which is not coat | covered with protein (n = 151 or n = 142). In FIG. 7a, “****” means p <0.0001, which is a result of comparison with a wild-type neuron by t-test under the same culture conditions. In FIG. 7b, the number of each sample is n = 600. In FIGS. 7a and b, error bars represent SEM.

As shown in FIGS. 7a and b, the neurite length of Dscam ^{− / −} derived DRG neurons on a Dscam-Fc coated petri dish is considerably shorter than that of wild type derived DRG neurons (FIG. 7a). We observed that the ratio of DRG neurons bearing neurites did not differ between Dscam ^{− / −} versus wild type DRG neurons (FIG. 7b). These results indicate that Dscam homophilic binding stimulates neurite outgrowth and Dscam deficiency can lead to the formation of abnormal neural networks.

These indicate that the expression of Dscam in RVLM can play an important role in the formation of its internal network (eg, axonal guidance receptors), and that its deficiency in Dscam ^{− / −} mice It has been shown that it leads to a type of abnormal internal connection, which can result in a loss of pre-I neuronal activity as a whole.

[Example 3] Analysis of the DSCAM gene From Examples 1 and 2, it was revealed that the DSCAM gene is involved in central respiratory disorder. Diagnosis of central respiratory disorder by detecting mutations in the DSCAM gene Can help. In this example, a heteroduplex method combined with a DHPLC method will be described as one method for detecting a mutation in the DSCAM gene.

In this method, first, an exon of the DSCAM gene was amplified by PCR. For PCR, genomic DNA was extracted from heparinized blood using QIAamp DNA Blood Mini Kit (Qiagen, manufactured by Hilden). PCR uses 33 pairs of primer sets designed to amplify 33 exons of the DSCAM gene and Pyrobest ^TM DNA polymerase (Takara) or Pwo DNA polymerase (Roche) using the extracted genomic DNA as a template did. Tables 1 and 2 show the base sequences of the 33 pairs of primer sets used in this example. For 33 exons in the DSCAM gene, see “Yamakawa et al.“ DSCAM: a novel member of the immunoglobulin superfamily maps in a Down syndrome region and is involved in the development of the nervous system. ”Hum. Mol. Genet. 7, 227-237 (1998).

For PCR to amplify exons 2 to 33, 50 to 100 ng of genomic DNA as a template is used, each primer is 0.5 μM, 1 × Pyrobest ^™ buffer and dNTP mixed solution is 0.2 mM and Pyrobest ^™ DNA polymerase is contained in a total volume of 25 μl. The reaction solution was used. In PCR for amplifying exon 1, 2.5 U of Epicentre MasterAmp KN buffer and Pwo DNA polymerase were used. PCR conditions for amplifying exons 2 to 33 are 3 cycles of initial denaturation at 95 ° C for 3 minutes followed by 1 cycle of 94 ° C for 20 seconds and 68 ° C for 1 minute. The temperature is then set to decrease by 1 ° C at a time, followed by 13 cycles of 94 ° C for 20 seconds, 64.5 ° C for 20 seconds, and 72 ° C for 40 seconds (however, at 64.5 ° C every cycle) Set the temperature of the step to decrease by 0.8 ° C for 20 seconds), and then perform 19 cycles of 94 ° C for 20 seconds, 55 ° C for 20 seconds and 72 ° C for 40 seconds, then 72 ° C The final elongation for 5 minutes was used. PCR conditions for amplifying exon 1 are 10 cycles (1 cycle at 94 ° C for 2 minutes, followed by 94 ° C for 15 seconds, 62 ° C for 30 seconds and 72 ° C for 1 minute) Set the temperature of the step at “62 ° C. for 30 seconds” to decrease by 1 ° C.), then 23 cycles of 94 ° C. for 15 seconds, 52 ° C. for 30 seconds and 72 ° C. for 1 minute. After that, the conditions were such that final extension at 72 ° C. for 5 minutes was performed. PCR confirmed the amplified product on a 2% agarose gel and showed that all exons 1-33 were able to amplify at the expected size (data not shown). The obtained PCR product was purified using an ArrayIt ^™ PCR purification kit (manufactured by TeleChem International) and subjected to sequencing. Then, sequencing was performed using BigDye ^™ terminator cycle sequencing kit and ABI PRISM ^™ 3700 DNA sequencing system (PE Applied Biosystems).

  Next, in this method, the above-mentioned PCR product was thermally denatured by treating at 95 ° C. for 5 minutes, and then treated at 80 to 55 ° C. for 50 minutes to perform stepwise reannealing. Thereby, about exons 1-33, a heteroduplex can be formed.

  Next, heteroduplex was detected by DHPLC method using the reaction solution. The DHPLC method was performed using a WAVE DNA-fragment analysis system (manufactured by Transgenomic). At this time, the column temperature was determined based on WAVEMAKER software (Transgenomic). Further, the reaction solution subjected to the DHPLC method did not have a polymorphism, and an equal amount of a control that formed a homoduplex and showed a single peak was added. Further, the reaction solution to which the control was added was eluted with a linear acetonitrile concentration gradient at a rate of 0.9 ml / min.

  Specific temperature conditions are 59 ° C. and 60 ° C. for exons 2, 21, and 22, and 60 ° C. and 61 ° C. for exons 3, 6, 8, 15, 16, 19, and 27, and exons 4, 7 , 10, 11, 12, 14, 17, 18, 24, 25 and 29 are 58 ° C and 59 ° C, exons 5, 20 and 26 are 62 ° C and 63 ° C, and exons 9, 30 and 32 are 61 ° C and 62 ° C, exon 13 at 56 ° C and 57 ° C, exon 23 and 28 at 54 ° C and 55 ° C, exon 31 at 63 ° C and 64 ° C, and exon 33 at 55 ° C and 56 ° C It was.

  Data analysis in the DHPLC method was performed by visual inspection on the obtained chromatogram. Each sample was also compared with the control. Heterozygous and homozygous mutation profiles were detected as peaks distinguishable from wild type homozygous peaks.

Schematic diagram for explaining the procedure for producing DSCAM gene knockout mice, photographs showing the results of Southern blot analysis for detecting DSCAM gene in the produced knockout mice and wild type, Dscam ^+/- and Dscam ^-/- mice It is a photograph which shows the result of the western blot analysis of RNA extracted from the brain of the. It is a photograph of the sagittal section of the P0 wild type mouse brain hybridized with the Dscam antisense probe. Photo of the ventral side of Dscam ^-/- and wild-type mice on day 0 after birth, characteristic diagram showing the results of measuring the width of the cerebrum and medulla, and photos after nissel staining of medullary and spinal cord sections , And photographs after sagital direction continuous sections derived from wild type and Dscam ^{− / −} mice. Dscam ^-/- , Dscam ^+/- Characteristic diagram showing the results of measuring respiratory rhythm using whole-body plethysmography for pups and wild-type littermates, and whole-cell patch clamp for membrane potential of inspiratory neurons It is a characteristic view which shows the result measured using the method. It is the photograph and characteristic figure which show the result measured by the optical recording system using the brainstem-spinal cord preparation dye | stained with the voltage-sensitive dye. It is a characteristic figure which shows the result of having measured the membrane potential of several neurons in the corresponding | compatible area | region of RVLM derived from a wild type mouse | mouth using the whole cell patch clamp method. It is a characteristic view which shows the result of a neurite outgrowth assay.

Claims (11)

  A non-human animal having a genotype deficient in the Down syndrome cell adhesion molecule (DSCAM) gene and exhibiting central respiratory disorder.   The non-human animal according to claim 1, wherein the DSCAM gene is homo-deficient.   The non-human animal according to claim 1, wherein the DSCAM gene is hetero-deficient. A method for producing a non-human animal having a genotype deficient in a Down syndrome cell adhesion molecule (DSCAM) gene and exhibiting central respiratory disorder,
Selecting an embryonic stem cell in which the gene in the non-human animal embryonic stem cell is deleted or rendered non-functional;
A chimeric embryo is produced from the embryonic stem cell,
Transplanting the chimeric embryo into the uterus of a temporary parent non-human animal, obtaining a non-human pup by birth, selecting the chimeric non-human animal in which the gene is disrupted,
By mating female and male of the chimeric non-human animal, a non-human animal having a genotype deficient in the DSCAM gene is obtained.
A method comprising steps.   The method according to claim 4, wherein a non-human animal in which the DSCAM gene is homo-deficient is obtained by the mating.   The method according to claim 4, wherein the mating yields a non-human animal in which the DSCAM gene is heterozygous. Administering a candidate substance to the non-human animal according to any one of claims 1 to 3,
Measuring at least one data of (1) to (4) above;
(1) Data indicating respiratory rhythm of non-human animals (2) Data indicating C4 anterior root activity (3) Data indicating anterior respiratory neuron activity (4) Data indicating neurite outgrowth of dorsal root ganglion neuron culture If any one or more of the following (1) to (4) is significantly improved as compared to the data measured using the non-human animal not administered with the candidate substance, the candidate substance is treated as a respiratory center disorder. And a method for screening a therapeutic agent for a disease caused by respiratory center disorder, comprising the step of identifying the therapeutic agent for a disease caused by the disease.   The screening method according to claim 7, wherein the disease is congenital central hypoventilation syndrome.   The screening method according to claim 7, wherein the disease is accompanied by Hirschspring disease. Collecting a biological sample from the subject,
A method for examining a disease caused by a respiratory center disorder, comprising a step of detecting a mutation in a Down syndrome cell adhesion molecule (DSCAM) gene in a collected biological sample.   A kit for diagnosing a disease caused by respiratory center disorder, comprising a reagent for detecting a mutation in a Down syndrome cell adhesion molecule (DSCAM) gene in a biological sample collected from a subject.

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