JP2010246547A - Mouse deleted in hematopoietic prostaglandin d synthetase gene and examination method using the same - Google Patents
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Abstract
Description
この発明は、遺伝子欠損マウスと、この遺伝子欠損マウスを用いた各種試験方法に関するものである。さらに詳しくは、この発明は、造血器型プロスタグランジンＤ合成酵素（Ｈ−ＰＧＤＳ）をコードする遺伝子がノックアウト（機能破壊）されており、この合成酵素の欠損によって免疫系機能、中枢神経系機能、循環器系機能および生殖機能等に、先天性または反応性の障害を呈する遺伝子欠損マウスと、この遺伝子欠損マウスを用いて上記機能障害に対する予防または治療薬剤の有効成分を試験する方法に関するものである。 The present invention relates to a gene-deficient mouse and various test methods using the gene-deficient mouse. More specifically, in the present invention, a gene encoding a hematopoietic prostaglandin D synthase (H-PGDS) is knocked out (function disrupted). , Gene-deficient mice exhibiting congenital or responsive disorders in cardiovascular function and reproductive function, etc., and methods for testing active ingredients of preventive or therapeutic drugs for the above-mentioned functional disorders using the gene-deficient mice. is there.
Ｈ−ＰＧＤＳ（非特許文献１、非特許文献２、非特許文献３等参照）は、各種の生理活性を有する体内物質プロスタグランジンＤ２（ＰＧＤ２：非特許文献４、非特許文献５、非特許文献６等参照）の産生機能を有する酵素であり、免疫担当細胞、生殖器官（非特許文献７、非特許文献８、非特許文献９等参照）に発現する。Ｈ−ＰＧＤＳにより肥満細胞から産生されるＰＧＤ２が炎症の増悪に関与することや、ＰＧＤ２の分解物質である１５ｄ−ＰＧＪ２が脂肪細胞の分化因子であることが知られている。
H-PGDS (see Non-Patent
Ｈ−ＰＧＤＳは肥満細胞、抗原提示細胞あるいはＴｈ２細胞に存在し（非特許文献１０、非特許文献１１、非特許文献１２等参照）、アレルギーを始めとした各種炎症反応におけるＰＧＤ２の産生に関わっている。これらの細胞から産生されたＰＧＤ２は、２種類の受容体を介して様々な生理活性を示す。アレルギー反応においては気管支収縮、血管拡張作用によって気道狭窄をおこし（非特許文献１３、非特許文献１４等参照）さらには好酸球を始めとした炎症細胞を局所に集積させ病態の増悪に働く（非特許文献１５、非特許文献１６、非特許文献１７等参照）。
H-PGDS is present in mast cells, antigen-presenting cells, or Th2 cells (see Non-Patent
また、ＰＧＤ２は現在までに明らかにされている内因性睡眠誘発物質のうちで最も強い催眠作用を示す。ヒトにおいてトリパノソーマ感染によるアフリカ睡眠病患者において、病状の進行に伴い脳脊髄液中のＰＧＤ２レベルが１００−１，０００倍上昇することが報告されている（非特許文献１８等参照）。さらに全身性肥満細胞増多症の患者に見られる病理的な深い眠りにおいても血液中のＰＧＤ２のレベルが１５０倍も上昇することが知られており（非特許文献１９等参照）、病的睡眠におけるＰＧＤ２の重要な役割が示唆されている。 PGD 2 exhibits the strongest hypnotic action among endogenous sleep inducers that have been clarified so far. It has been reported that PGD 2 level in cerebrospinal fluid increases 100 to 1,000 times as the disease progresses in African sleep disease patients due to trypanosoma infection in humans (see Non-Patent Document 18 etc.). It is also known that the level of PGD 2 in the blood also increases 150-fold in more pathological deep sleep seen in patients with systemic mast cell sensitization multi syndrome (see Non-Patent Document 19, etc.), pathological An important role for PGD 2 in sleep has been suggested.
また、中枢神経系でのＰＧＤ２の増減は、侵害的な刺激（例えば痛覚刺激）に対する感受性の変化にも関与することが知られている（非特許文献２０、非特許文献２１、非特許文献２２等参照）。
さらに、生殖器官においてＨ−ＰＧＤＳは、卵管に特異的に発現し（非特許文献２３等参照）、生殖機能の調節に関係していること示唆される。
ＰＧＤ２は血小板凝集抑制作用を示すことも知られている。
In addition, it is known that the increase or decrease of PGD 2 in the central nervous system is also involved in changes in sensitivity to noxious stimuli (for example, painful stimuli) (Non-patent
Furthermore, H-PGDS is specifically expressed in the oviduct (see Non-Patent Document 23 etc.) in the reproductive organs, suggesting that it is involved in the regulation of reproductive function.
PGD 2 is also known to exhibit a platelet aggregation inhibitory action.
以上のとおり、ＰＧＤ２とこれを産生するＨ−ＰＧＤＳが生物個体の様々な生理機能に密接に関係しており、その欠損が種々のヒト疾患要因となりうることが示唆されている。
しかしながら、Ｈ−ＰＧＤＳ活性の欠損が動物個体に対してどの様に作用するのかについて、統制された条件下での研究を可能とするモデル動物系は確立されていない。
As described above, PGD 2 and H-PGDS that produces it are closely related to various physiological functions of living organisms, and it has been suggested that the deficiency can cause various human diseases.
However, no model animal system has been established that allows studies under controlled conditions on how the deficiency of H-PGDS activity acts on individual animals.
また、そのようなモデル動物は、Ｈ−ＰＧＤＳ活性の欠損によって生じる各種疾患の予防もしくは治療薬剤の開発等にも極めて有効であろうと期待される。
この出願の発明は、以上のとおりの事情に鑑みてなされたものであって、遺伝的にＨ−ＰＧＤＳ活性を欠損しているマウス個体を提供することを目的としている。またこの出願は、このマウス個体を用い、Ｈ−ＰＧＤＳ活性の欠損によって生じる各種疾患の予防もしくは治療物質の有効性を試験する方法を提供することを目的としてもいる。
In addition, such a model animal is expected to be extremely effective for the prevention or development of therapeutic agents for various diseases caused by a deficiency in H-PGDS activity.
The invention of this application has been made in view of the circumstances as described above, and an object thereof is to provide a mouse individual that is genetically deficient in H-PGDS activity. Another object of the present application is to provide a method for testing the effectiveness of a prophylactic or therapeutic substance for various diseases caused by deficiency in H-PGDS activity using this mouse individual.
この出願は、上記の課題を解決するための発明として、ゲノムＤＮＡのＨ−ＰＧＤＳ遺伝子が、そのエクソンＩＩがネオマイシン耐性遺伝子に置換された変異配列に相同組換えされたマウス胚性幹細胞を導入した初期胚を雌マウス体内で個体へと発生させて産生させたキメラマウスの子孫個体であって、生殖細胞および体細胞ゲノムＤＮＡのＨ−ＰＧＤＳ遺伝子が変異配列に置換されている遺伝子を提供する。 As an invention for solving the above-mentioned problems, this application introduces mouse embryonic stem cells in which H-PGDS gene of genomic DNA is homologously recombined with a mutant sequence in which exon II is replaced with neomycin resistance gene. Provided is a progeny individual of a chimeric mouse produced by generating an early embryo into an individual in a female mouse, wherein the H-PGDS gene in germ cell and somatic cell genomic DNA is replaced with a mutant sequence.
なお、この遺伝子欠損マウスは、Ｈ−ＰＧＤＳの対立遺伝子の両方または片方が変異配列に置換されていることを好ましい態様の一つとしている。 One preferred embodiment of this gene-deficient mouse is that both or one of the H-PGDS alleles is replaced with a mutant sequence.
さらにこの出願は、以下の各種試験方法を提供する。
（１）アレルギーを調節する物質の個体内活性を試験する方法であって、上記の遺伝子欠損マウスに候補物質を投与し、このマウスのアレルギー反応を測定することを特徴とする試験方法。
（２）急性炎症を引き起こす物質の個体内活性を試験する方法であって、上記の遺伝子欠損マウスに候補物質を投与し、このマウスの急性炎症反応を測定することを特徴とする試験方法。
（３）肥満および脂肪細胞分化調節物質の個体内活性を試験する方法であって、上記の遺伝子欠損マウスに候補物質を投与し、このマウスの肥満状態を測定することを特徴とする試験方法。
（４）組織傷害を調節する物質の個体内活性を試験する方法であって、上記の遺伝子欠損マウスに候補物質を投与し、このマウスの組織の損傷を測定することを特徴とする試験方法。
（５）睡眠調節物質の個体内活性を試験する方法であって、上記の遺伝子欠損マウスに候補物質を投与し、このマウスの睡眠状態を測定することを特徴とする試験方法。
（６）鎮痛物質の個体内活性を試験する方法であって、上記の遺伝子欠損マウスに候補物質を投与し、このマウスの痛覚刺激に対する反応性を測定することを特徴とする試験方法。
（７）血液凝固抑制物質の個体内活性を試験する方法であって、上記の遺伝子欠損マウスに候補物質を投与し、このマウスの血液凝固の程度を測定することを特徴とする試験方法。
（８）不妊改善物質の個体内活性を試験する方法であって、上記の遺伝子欠損マウスの雌に、妊娠前または妊娠後に候補物質を投与し、この雌マウスの子宮における受精卵の着床状態または胎仔の発育状態を測定することを特徴とする試験方法。
（９）麻酔物質の個体内活性を試験する方法であって、上記の遺伝子欠損マウスに候補物質を投与し、このマウスの麻酔状態および／または麻酔からの覚醒状態を測定することを特徴とする試験方法。
（１０）生理不順改善物質の個体内活性を試験する方法であって、上記の遺伝子欠損マウスの雌に候補物質を投与し、この雌マウスの性周期、排卵数および／または各種ホルモン量を測定することを特徴とする試験方法。
Furthermore, this application provides the following various test methods.
(1) A method for testing the activity within a substance of a substance that regulates allergy, comprising administering a candidate substance to the gene-deficient mouse and measuring the allergic reaction of the mouse.
(2) A method for testing the activity of a substance causing acute inflammation in an individual, wherein the candidate substance is administered to the gene-deficient mouse, and the acute inflammatory reaction of the mouse is measured.
(3) A method for testing the intra-individual activity of a substance that regulates obesity and adipocyte differentiation, comprising administering a candidate substance to the gene-deficient mouse and measuring the obesity state of the mouse.
(4) A method for testing the intra-individual activity of a substance that regulates tissue injury, comprising administering a candidate substance to the gene-deficient mouse and measuring the tissue damage of the mouse.
(5) A method for testing the activity of a sleep regulator in an individual, the method comprising administering a candidate substance to the gene-deficient mouse and measuring the sleep state of the mouse.
(6) A method for testing the intra-individual activity of an analgesic substance, which comprises administering a candidate substance to the above gene-deficient mouse and measuring the reactivity of the mouse to painful stimuli.
(7) A method for testing the activity of a blood coagulation inhibitor in an individual, the method comprising administering a candidate substance to the gene-deficient mouse and measuring the degree of blood coagulation in the mouse.
(8) A method for testing the in-individual activity of an infertility improving substance, wherein a candidate substance is administered to a female of the above gene-deficient mouse before or after pregnancy, and the implantation state of a fertilized egg in the uterus of this female mouse Or the test method characterized by measuring the growth state of a fetus.
(9) A method for testing the activity of an anesthetic substance in an individual, wherein the candidate substance is administered to the gene-deficient mouse, and the anesthesia state and / or the arousal state from the anesthesia is measured. Test method.
(10) A method for testing the intra-individual activity of a physiological disorder-improving substance, wherein a candidate substance is administered to females of the above gene-deficient mice, and the sexual cycle, the number of ovulations and / or the amounts of various hormones of the female mice are measured. A test method characterized by:
この発明によって、Ｈ−ＰＧＤＳ遺伝子がノックアウトされており、この合成酵素の欠損によって免疫系機能、中枢神経系機能、循環器系機能および生殖機能等に、先天性または反応性の障害を呈する遺伝子欠損マウスが提供される。この遺伝子欠損マウスを用いることによって、上記の機能障害に対する予防または治療薬剤の有効成分を動物個体レベルで試験することが可能となる。 According to the present invention, the H-PGDS gene has been knocked out, and the gene defect presenting a congenital or reactive disorder in immune system function, central nervous system function, circulatory system function, reproductive function, etc. due to the deficiency of this synthetic enzyme A mouse is provided. By using this gene-deficient mouse, it becomes possible to test the active ingredient of the preventive or therapeutic agent for the above-mentioned functional disorder at the animal individual level.
この発明の遺伝子欠損マウスは、公知の標的遺伝子組換え法（ジーン・ターゲティング：例えば、Ｍethods in Ｅnzymology ２２５：８０３−８９０，１９９３）を用いることにより、例えば以下のとおりに作製することができる。
先ず、単離したＨ−ＰＧＤＳ遺伝子の配列中のエクソンＩＩをネオマイシン耐性遺伝子（Ｎｅｏｒ遺伝子）と置換し、またＨ−ＰＧＤＳ遺伝子の端部にヘルペスウイルスのサイミジンカイネース遺伝子（ＨＳＶ−ｔｋ遺伝子）を付加してターゲティング・ベクターを作製する。このターゲティング・ベクターをマウスの胚性幹細胞（ＥＳ細胞）に導入し、細胞ゲノムＤＮＡのＨ−ＰＧＤＳ遺伝子がターゲティング・ベクター中の変異配列に相同組換えされた細胞を選択する。このような遺伝子組換え細胞の選択は、Ｇ４１８を細胞培地に添加してＮｅｏｒ遺伝子を持たない非組換え細胞を除去し、さらにガンシクロビルを添加してＨＳＶ−ｔｋ遺伝子が残存するランダムな組換え細胞を除去することによって行うことができる。選択された遺伝子組換え細胞のＨ−ＰＧＤＳ遺伝子は、そのコード配列中にＮｅｏｒ遺伝子が挿入された変異配列であり、Ｈ−ＰＧＤＳを産生することはできない。
The gene-deficient mouse of the present invention can be prepared, for example, as follows by using a known target gene recombination method (gene targeting: for example, Methods in Enzymology 225: 803-890, 1993).
First, thymidine kinase scan gene H-PGDS exon II in the sequence of the gene was replaced with the neomycin resistance gene (Neo r gene), also herpesvirus an end portion of the H-PGDS gene isolated (HSV-tk gene ) To create a targeting vector. This targeting vector is introduced into mouse embryonic stem cells (ES cells), and cells in which the H-PGDS gene of the cell genomic DNA is homologously recombined with the mutant sequence in the targeting vector are selected. The selection of such genetically modified cells, random recombination of G418 added to the cell culture medium to remove non-recombinant cells without Neo r gene, HSV-tk gene further added ganciclovir remain This can be done by removing the cells. H-PGDS gene selected genetically modified cells, the coding sequence in a mutant sequence Neo r gene inserted, it is not possible to produce H-PGDS.
次いで、この遺伝子組換えされたＥＳ細胞をマウスの初期胚（胚盤胞）に注入し、この初期胚を雌マウスの体内で個体へと発生させ、キメラマウスを産生させる。そして、このキメラマウスと野生型マウスとを交配して子孫マウスを産生させ、これらの子孫マウスの中から、対立遺伝子の両方または片方に変異配列を有するマウス個体を選別することによって、この発明の遺伝子欠損マウスを得ることができる。Ｈ−ＰＧＤＳ産生能を持たない、またはＨ−ＰＧＤＳ産生量が野生型に比べて低い遺伝子欠損マウスを作製することができる。なお、後記する実施例にも示したように、対立遺伝子の両方が変異配列に置換されている遺伝子欠損マウス（ホモ接合体：−／−）は、野生型マウス（＋／＋）に比べＨ−ＰＧＤＳ活性は１０％以下である。また、対立遺伝子の片方が変異配列に置換されている遺伝子欠損マウス（ヘテロ接合体：＋／−）はＨ−ＰＧＤＳ産生量が野生型マウス（＋／＋）の約半分である。 Next, this genetically modified ES cell is injected into an early embryo (blastocyst) of a mouse, and this early embryo is developed into an individual in a female mouse to produce a chimeric mouse. Then, the chimeric mouse and the wild-type mouse are crossed to produce offspring mice, and from these offspring mice, a mouse individual having a mutated sequence in both or one of the alleles is selected. Gene-deficient mice can be obtained. A gene-deficient mouse that does not have the ability to produce H-PGDS or has a lower H-PGDS production amount than the wild type can be produced. In addition, as shown also in the Example mentioned later, the gene-deficient mouse | mouth (homozygote:-/-) by which both alleles were substituted by the variation | mutation sequence is H compared with a wild type mouse | mouth (+ / +). -PGDS activity is 10% or less. In addition, gene-deficient mice (heterozygote: +/−) in which one of the alleles is replaced with a mutant sequence have about half the amount of H-PGDS produced as compared to wild-type mice (+ / +).
このようにして作成した遺伝子欠損マウスは、以下のとおりの試験方法に用いることができる。 The gene-deficient mouse thus prepared can be used in the following test method.
（１）アレルギー調節物質の試験方法
野生型マウス等に任意のアレルゲンを投与し、Ｈ−ＰＧＤＳ活性阻害剤を投与すると、非投与マウスに比べてアレルギー病態が軽微であるが、この発明の遺伝子欠損マウスは遺伝形質として、アレルギー病態が軽微である。そこで、アレルギー調節物質の候補物質を遺伝子欠損マウスに投与し、マウスのアレルギー状態を測定することによって、例えばアレルギー調節物質の個体内活性を試験することができる。さらに、アレルギーにおけるＨ−ＰＧＤＳの重要性を評価することが可能になる。なお、マウスのアレルギー病態の測定は、例えば気道収縮等の呼吸機能、炎症細胞の局所への集積、皮膚の浮腫、血圧、引っ掻き行動等を経時的に測定することによって行うことができる。
(1) Test method for allergic regulators When an allergen is administered to a wild-type mouse or the like and an H-PGDS activity inhibitor is administered, the allergic pathology is milder than that of a non-administered mouse. Mice have a mild allergic condition as a genetic trait. Therefore, by administering a candidate substance for an allergic regulator to a gene-deficient mouse and measuring the allergic state of the mouse, for example, the activity of the allergic regulator in the individual can be tested. Furthermore, it becomes possible to evaluate the importance of H-PGDS in allergy. The allergic pathology of mice can be measured by measuring, for example, respiratory function such as airway contraction, local accumulation of inflammatory cells, skin edema, blood pressure, scratching behavior and the like over time.
（２）急性炎症調節物質の試験方法
野生型マウス等にエンドトキシンなどの微生物由来の毒素を投与すると、例えば呼吸器においては顕著な好中球の集積を特徴とした急性炎症が認められるが、この発明の遺伝子欠損マウスは遺伝形質として、急性炎症が重篤である。そこで、例えば事前に任意の微生物由来毒素を遺伝子欠損マウスに投与し、次いで、抗炎症薬等の候補物質を投与して、動物の炎症状態を測定することによって、候補物質の薬効を評価することができる。
(2) Acute inflammation regulator test method When a toxin derived from a microorganism such as endotoxin is administered to a wild-type mouse or the like, for example, acute inflammation characterized by marked neutrophil accumulation is observed in the respiratory tract. The gene-deficient mice of the invention have severe acute inflammation as a genetic trait. Therefore, for example, by administering an arbitrary microorganism-derived toxin to a gene-deficient mouse in advance, then administering a candidate substance such as an anti-inflammatory drug, and measuring the inflammatory state of the animal, to evaluate the efficacy of the candidate substance Can do.
（３）肥満および脂肪細胞分化調節物質の試験方法
この発明の遺伝子欠損マウスに肥満および脂肪細胞分化調節候補物質を投与し、この動物の肥満の程度（体重、脂肪組織重量、血液中の中性脂肪など）を測定することによって、候補物質の個体内活性を試験することができる。すなわち、遺伝子欠損マウスは、Ｈ−ＰＧＤＳをほとんど、または半分程度しか発現しないため、それによって合成されるＰＧＤ２やその代謝物である１５ｄ−ＰＧＪ２（１５−deoxyΔ１２，１４ＰＧＪ２）の産生量が少ない。１５ｄ−ＰＧＪ２は体重増加や脂肪組織重量の増加に関与する物質であり、この動物は肥満よび脂肪細胞分化に異常が認められる。そこで、肥満および脂肪細胞分化調節候補物質を投与して、動物の肥満状態を測定することによって、候補物質の個体内活性を試験することができる。さらに、肥満の発症および脂肪細胞分化機構の解明にも利用することができる。
(3) Test method for obesity and adipocyte differentiation regulator Substances of obesity and adipocyte differentiation regulator are administered to the gene-deficient mice of this invention, and the degree of obesity (weight, adipose tissue weight, neutrality in blood) of this animal The individual activity of the candidate substance can be tested by measuring fat etc.). That is, since a gene-deficient mouse expresses almost or only half of H-PGDS, the production amount of PGD 2 synthesized thereby and 15d-PGJ 2 (15-deoxyΔ12, 14PGJ 2 ), which is a metabolite thereof, is high. Few. 15d-PGJ 2 is a substance involved in weight gain and adipose tissue weight, and this animal has abnormal obesity and adipocyte differentiation. Therefore, by administering a candidate substance for regulating obesity and adipocyte differentiation and measuring the obesity state of the animal, the in vivo activity of the candidate substance can be tested. Furthermore, it can be used to elucidate the development of obesity and the mechanism of adipocyte differentiation.
（４）組織傷害調節物質の試験方法
この発明の遺伝子欠損マウスに組織傷害調節候補物質を投与して、この動物の組織傷害からの治癒の程度を測定することによって、候補物質の個体内活性を試験する方法である。すなわち、この発明の遺伝子欠損マウスに、人為的操作（外科的操作）によって組織傷害を起こすと野生型マウス等に比べて、治癒が早いことを特徴としている。従って、例えば、事前に人為的操作によって組織傷害を与え、次いで候補物質を投与して、治癒の程度を経時的に測定することによって、候補物質の個体内活性を評価することができる。
(4) Test Method for Tissue Injury Modulating Substance A candidate substance for tissue injury regulation is administered to the gene-deficient mouse of the present invention, and the degree of healing from tissue injury in this animal is measured, whereby the in vivo activity of the candidate substance is determined. How to test. That is, the gene-deficient mouse of the present invention is characterized in that healing is quicker than that of a wild-type mouse or the like when tissue damage is caused by an artificial operation (surgical operation). Therefore, for example, the in vivo activity of the candidate substance can be evaluated by giving tissue damage in advance by an artificial operation, then administering the candidate substance, and measuring the degree of healing over time.
（５）睡眠調節物質の試験方法
野生型マウス等にＨ−ＰＧＤＳ活性阻害剤を脳室内投与すると、顕著な睡眠障害が認められるが、この発明の遺伝子欠損マウスは遺伝形質としての睡眠障害を呈する。そこで、催眠薬等の有効成分となる睡眠調節物質の候補物質を遺伝子欠損マウスに投与し、マウスの睡眠状態を測定することによって、例えば副作用の少ない睡眠調節物質の探索、そのような物質を有効成分とする催眠薬の開発が可能となる。また、このような睡眠調節物質の探索等をとおして、哺乳動物における睡眠調節機構の解明も可能となる。なお、マウスの睡眠状態の測定は、例えば脳波、筋電、活動量、摂食・摂水量、体温等を経時的に計測することによって行うことができる。
(5) Test method for sleep regulator substance When a H-PGDS activity inhibitor is administered into the ventricle in a wild-type mouse or the like, a remarkable sleep disorder is observed, but the gene-deficient mouse of this invention exhibits a sleep disorder as a genetic trait. . Thus, by administering a candidate substance of a sleep regulator as an active ingredient such as a hypnotic to a gene-deficient mouse and measuring the sleep state of the mouse, for example, searching for a sleep regulator with fewer side effects, such a substance is effective. Development of hypnotics as ingredients is possible. Moreover, elucidation of the sleep regulatory mechanism in mammals becomes possible through the search for such a sleep regulatory substance. The sleep state of the mouse can be measured by measuring, for example, electroencephalogram, myoelectricity, activity, food intake / water intake, body temperature, etc. over time.
（６）鎮痛物質の試験方法
ヒトは健康時には接触刺激によって痛みを感じることはないが、例えば帯状疱疹に罹患した場合のような病的状況下では軽い触覚刺激でも激痛を起こす。これはアロディニアと呼ばれる現象であり、熱刺激や機械刺激による痛覚過敏とは区別されている（Ｔextbook of Ｐain．３rd Ｅd，ｐｐ１６５−２００，１９９４；Ｐain ６８：１３−２３，１９９６）。この発明の遺伝子欠損マウスは、人為的操作によるアロディニアを全く示さず、熱刺激に対する痛覚過敏症状を示すことを特徴としている。
従って、この発明の遺伝子欠損マウスは、アロディニアおよび痛覚過敏誘発機構の解明に利用することができる。また、鎮痛物質の候補をマウスに投与し、このマウスの痛覚刺激に対する反応性を測定することによって、痛覚反応に選択的な鎮痛薬を開発することが可能となる。さらに、痛覚誘発機構の解明と新規な鎮痛薬の開発は、例えばモルヒネで抑えることのできないガン末期の激痛メカニズムを解明し、その対処療法を開発するにも有効である。
(6) Test method for analgesic substances Although humans do not feel pain due to contact stimulation during health, they cause severe pain even with mild tactile stimulation under pathological conditions such as when suffering from shingles. This is a phenomenon called allodynia, and is distinguished from hyperalgesia caused by thermal stimulation or mechanical stimulation (Textbook of Pain. 3rd Ed, pp165-200, 1994; Pain 68: 13-23, 1996). The gene-deficient mouse of the present invention is characterized in that it does not show any allodynia caused by artificial manipulation and exhibits hyperalgesia to heat stimulation.
Therefore, the gene-deficient mouse of the present invention can be used for elucidation of allodynia and hyperalgesia-inducing mechanism. In addition, it is possible to develop an analgesic drug selective for pain reaction by administering a candidate analgesic substance to a mouse and measuring the reactivity of the mouse to pain stimulation. Furthermore, elucidation of the mechanism of pain induction and the development of new analgesics are also effective in elucidating the mechanism of severe pain at the end of cancer that cannot be suppressed by, for example, morphine, and developing coping therapy.
（７）血液凝固抑制物質の試験方法
従来、動脈硬化モデル動物としては、強制的に動脈硬化を生じさせたラットやウサギが用いられてきたが、マウスはヒトによく似た摂食による動脈硬化を発症させることができる（Ａtheroscleosis ５７：６５−７３，１９８５）。ＰＧＤ２は、血液凝固抑制作用を有することが知られており、動脈硬化による血管閉鎖を防止する。この発明の遺伝子欠損マウスは、Ｈ−ＰＧＤＳをほとんど、または半分程度しか発現しないため、それによって合成されるＰＧＤ２が少ないために、より顕著な形で動脈硬化症状を発症させることができる。
従って、動脈硬化を発症させた遺伝子欠損マウスに血液凝固抑制物質の候補を投与し、このマウスの動脈硬化の程度を測定することによって、ヒトの動脈硬化発症機構の解明とともに、新しい血液凝固抑制薬を開発することが可能である。
(7) Test method for blood coagulation-inhibiting substances Conventionally, rats and rabbits that have been forced to cause arteriosclerosis have been used as arteriosclerosis model animals. Can be developed (Atheroscleosis 57: 65-73, 1985). PGD 2 is known to have a blood coagulation inhibitory effect and prevents blood vessel closure due to arteriosclerosis. Since the gene-deficient mouse of the present invention expresses almost or only half of H-PGDS, the amount of PGD 2 synthesized thereby is small, and therefore, it is possible to develop arteriosclerotic symptoms in a more prominent manner.
Therefore, by administering a blood coagulation inhibitor candidate to a gene-deficient mouse that has developed arteriosclerosis, and measuring the degree of arteriosclerosis in this mouse, along with elucidation of the onset mechanism of human arteriosclerosis, a new blood coagulation inhibitor Can be developed.
（８）女性要因による不妊改善物質の試験方法
野生型の雌マウスでは、卵管で特異的にＨ−ＰＧＤＳが発現するのに対し、この発明の遺伝子欠損マウスでは、分娩は正常ではあるが、妊娠期間の顕著な延長が認められる。従って、この遺伝子欠損マウスは、受精卵の着床不全や胎児の発育遅滞の優れた動物モデルであり、マウスの妊娠前または妊娠後に候補物質を投与し、この雌マウスの子宮における受精卵の着床状態または胎児の発育状態を測定することによって、女性要因による不妊を改善するための薬剤の開発が可能となる。
(8) Method for testing fertility-improving substance due to female factors In wild-type female mice, H-PGDS is specifically expressed in the oviduct, whereas in gene-deficient mice of this invention, delivery is normal, A significant prolongation of gestation is observed. Therefore, this gene-deficient mouse is an excellent animal model for implantation failure of fertilized eggs and fetal growth delay, and a candidate substance is administered before or after pregnancy of the mouse, and the implantation of the fertilized egg in the uterus of this female mouse is performed. By measuring the floor condition or fetal developmental status, it is possible to develop drugs to improve infertility due to female factors.
（９）麻酔物質の試験方法
外科手術等の際には吸入麻酔物質を用いて全身麻酔を行う場合があるが、理想的な麻酔とは低濃度の吸入麻酔剤による無痛・筋弛緩・軽い睡眠状態をいう。しかし、麻酔状態には、性別・年齢・体格・健康状態等に依存した個体差があり、麻酔のかかりにくい患者では高濃度の吸入麻酔剤の使用により死に至る場合もある。このため、吸入麻酔剤の濃度は慎重に決定される必要があるが、吸入麻酔物質の中枢神経系への作用機序がほとんど解明されていないために、医療現場では熟練医師の経験則に頼らざるを得ないのが現状である。この発明の遺伝子欠損マウスは、吸入麻酔物質による麻酔の深度が浅いため、麻酔下および／または覚醒後の状態を測定することによって、痛覚消失・筋弛緩・睡眠に至るメカニズムを各々分離して解析することが可能である。これにより、現在使用されている吸入麻酔物質のより詳細な薬効評価が可能となるばかりか、新規の吸入麻酔物質の開発も可能となる。なお、マウスの麻酔状態としては、例えば正向反射の消失、無痛、筋弛緩、睡眠状態等をそれぞれ常法に従って測定することができる。また、覚醒状態は、例えば正向反射の回復、痛覚反応、筋力の回復、覚醒を測定することができる。
(9) Test method for anesthetic substance General anesthesia may be performed using an inhalation anesthetic substance during surgery, etc., but ideal anesthesia is painlessness, muscle relaxation, and light sleep with a low concentration of an inhalation anesthetic. State. However, there are individual differences in anesthesia depending on sex, age, physique, health condition, etc., and in patients who are difficult to receive anesthesia, the use of a high concentration of an inhalation anesthetic may result in death. For this reason, the concentration of the inhalation anesthetic must be carefully determined, but the mechanism of action of the inhalation anesthetic substance on the central nervous system has not been elucidated. The current situation is unavoidable. Since the gene-deficient mice of this invention have a shallow depth of anesthesia with inhaled anesthetic substances, the mechanisms leading to analgesia, muscle relaxation, and sleep are separated and analyzed by measuring the state under anesthesia and / or after waking. Is possible. As a result, it is possible not only to conduct a more detailed evaluation of the efficacy of currently used inhalation anesthetic substances, but also to develop new inhalation anesthetic substances. In addition, as an anesthesia state of a mouse | mouth, the loss | disappearance of a direct reflex, painlessness, muscle relaxation, a sleep state etc. can each be measured in accordance with a conventional method. Moreover, the arousal state can measure, for example, recovery of forward reflexes, pain response, muscle strength recovery, and arousal.
（１０）生理不順改善物質の試験方法
排卵周期と睡眠および活動量が同調することが知られている（Ｇen．Ｃomp．Ｅndocrinol．７：１０−１７，１９９６；Ｐhysiol．Ｂehav．４９：１０７９−１０８４，１９９１；Ｂrain Ｒes．７３４：２７５−２８５，１９９６；Ｂrain Ｒes．８１１：９６−１０４，１９９８）。生活リズムの変調により生理不順が起こるが、ホルモンの分泌異常による生理不順は社会生活に支障をきたすような、例えば過剰睡眠等を引き起こす。この発明の遺伝子欠損マウスは、排卵期前後が延長された性周期を示し、その結果、野生型マウスに比べて性周期が長い。ＰＧＤ２は排卵誘発を促すホルモンの分泌量を変化させるため、この発明の遺伝子欠損マウスは、排卵の誘発機構および生理不順による過剰睡眠等の誘発機構を解明するために有効であり、新規の生理不順改善物質の開発が可能となる。
(10) Test method of physiological disorder improving substance It is known that the ovulation cycle, sleep and activity amount are synchronized (Gen. Comp. Endocrinol. 7: 10-17, 1996; Physiol. Behav. 49: 1079-1084). , 1991; Brain Res. 734: 275-285, 1996; Brain Res. 811: 96-104, 1998). Physiological irregularities occur due to the modulation of life rhythms, but irregular physiology due to abnormal secretion of hormones causes, for example, excessive sleep, which interferes with social life. The gene-deficient mouse of the present invention exhibits a sexual cycle that is extended before and after the ovulation period, and as a result, has a longer sexual cycle than wild-type mice. Since PGD 2 changes the secretion amount of hormones that induce ovulation induction, the gene-deficient mouse of the present invention is effective for elucidating the induction mechanism of ovulation and the induction mechanism such as excessive sleep due to physiological irregularity. Development of irregularly improved substances becomes possible.
以上の各試験方法においては、この発明の遺伝子欠損マウスのホモ接合型（−／−）およびヘテロ接合型（＋／−）を適宜に使い分けることによって、Ｈ−ＰＧＤＳ産生量に依存した各種症状と、それに対する試験物質の効果を詳細に分析することが可能である。 In each of the above test methods, by appropriately using the homozygous type (− / −) and the heterozygous type (+/−) of the gene-deficient mouse of the present invention, various symptoms depending on the amount of H-PGDS produced can be obtained. It is possible to analyze in detail the effect of the test substance on it.
以下、実施例を示してこの発明をさらに詳細に説明するが、この発明は以下の例に限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to the following examples.
実施例１（遺伝子欠損マウスの作製）
公知のマウスＨ−ＰＧＤＳ遺伝子のエクソンＩＩ（Ｈ−ＰＧＤＳの蛋白質翻訳開始領域）を含む領域をＮｅｏｒ遺伝子に置換し、さらにＨ−ＰＧＤＳ遺伝子の約７Ｋｂ上流にＨＳＶ−ｔｋ遺伝子を組み込んで変異配列を調製し、この変異配列をベクターに組み込んでターゲティング・ベクターを作製した（図１参照）。なお、図１にも示した様に、変異配列のＨ−ＰＧＤＳコード領域は約３．４ｋｂとなっている。
Example 1 (Generation of gene-deficient mice)
A region including exon II (protein translation initiation region of the H-PGDS) of known murine H-PGDS gene was replaced with Neo r gene, further mutated sequences incorporate HSV-tk gene to about 7Kb upstream of H-PGDS gene And the mutant sequence was incorporated into a vector to prepare a targeting vector (see FIG. 1). As shown in FIG. 1, the H-PGDS coding region of the mutant sequence is about 3.4 kb.
電気穿孔法により、未分化の培養ＥＳ細胞（１．２×１０７個）にターゲティング・ベクターを４８μｇ／ｍｌの割合で導入して遺伝子導入ＥＳ細胞を得た。これらの細胞をプレートに播き、２日後にＧ４１８およびガンシクロビルを培地に添加して更に７日間培養し、Ｇ４１８およびガンシクロビルに耐性を示すコロニーを得た。これらのコロニを個別に分離し、さらに培養したのち、ＤＮＡを抽出してサザンブロッティングにより相同組換えＥＳ細胞を選別した。 By electroporation, a targeting vector was introduced into undifferentiated cultured ES cells (1.2 × 10 7 cells) at a rate of 48 μg / ml to obtain gene-transferred ES cells. These cells were seeded on a plate, and after 2 days, G418 and ganciclovir were added to the medium and further cultured for 7 days to obtain colonies resistant to G418 and ganciclovir. After these colonies were individually separated and further cultured, DNA was extracted and homologous recombinant ES cells were selected by Southern blotting.
次いで、この相同組換えＥＳ細胞を、Ｃ５７ＢＬ／６系マウスの胚盤胞へ常法により注入し、仮親マウスへ移植して個体へと発生させた。
その結果、１０匹のキメラマウスを得た。得られたキメラマウスのうち、雄の個体と雌の野生型Ｃ５７ＢＬ／６系マウスとを交配させて初代（Ｆ１）マウスを得た。これらのＦ１マウスから、サザンブロット分析により２倍体染色体の一方に変異配列が確認された個体（♂、♀）を選別し、これらを交配させて第２世代（Ｆ２）マウスを得た。
Subsequently, this homologous recombinant ES cell was injected into a blastocyst of a C57BL / 6 strain mouse by a conventional method, transplanted to a temporary parent mouse, and allowed to develop into an individual.
As a result, 10 chimeric mice were obtained. Among the obtained chimeric mice, male individuals and female wild-type C57BL / 6 mice were mated to obtain primary (F 1 ) mice. These F 1 mice, Southern blot analysis by individual mutated sequence was confirmed in one of diploid chromosomes (♂, ♀) were selected to obtain these mated with second generation (F 2) Mouse .
最終的に、これらＦ２マウスから、サザンブロット分析により２倍体染色体の両方に変異配列が確認された個体（ホモ接合体）および片方に変異配列が確認された個体（ヘテロ接合体）を選別し、この発明の遺伝子欠損動物を作製した。
なお、Ｆ２マウスの野生型：ヘテロ接合体：ホモ接合体の比は約１：２：１であり、雌雄比は約１：１であった。ホモ接合体およびヘテロ接合体とも胎仔性致死は認められなかった。
Finally, individuals with homozygous mutations in both diploid chromosomes (homozygote) and individuals with heterologous mutations in one (heterozygote) were selected from these F 2 mice by Southern blot analysis. Thus, the gene-deficient animal of this invention was produced.
Incidentally, F 2 mice wild type: heterozygous ratio of homozygotes about 1: 1, sex ratio is about 1: 2 1. No fetal lethality was observed in either homozygotes or heterozygotes.
図２は、各マウスの尾部から抽出したＤＮＡのサザンブロット分析の結果であり、野生型（＋／＋）は６．２ｋｂ、ホモ欠損型（−／−）は３．４ｋｂ、ヘテロ欠損型（＋／−）はその両者の発現が確認された。
図３は、マウスの脳から抽出したｍＲＮＡのノーザンブロット分析の結果であり、Ｈ−ＰＧＤＳｍＲＮＡは野生型でのみ発現が認められた。
FIG. 2 shows the results of Southern blot analysis of DNA extracted from the tail of each mouse. The wild type (+ / +) is 6.2 kb, the homo-deficient type (− / −) is 3.4 kb, and the hetero-deficient type ( The expression of both was confirmed in (+/−).
FIG. 3 shows the results of Northern blot analysis of mRNA extracted from the mouse brain. H-PGDS mRNA was expressed only in the wild type.
図４は、各マウスの卵管より抽出したＨ−ＰＧＤＳの酵素活性の分析結果である。ホモ欠損型マウスのＨ−ＰＧＤＳ活性は、野生型マウスの１０％以下、ヘテロ欠損型では約５０％であった。 FIG. 4 shows the analysis results of the enzyme activity of H-PGDS extracted from the oviduct of each mouse. H-PGDS activity of homo-deficient mice was 10% or less of wild-type mice, and about 50% of hetero-deficient mice.
実施例２（遺伝子欠損マウスのアレルギー性気道炎症の測定）
実施例１で得た遺伝子欠損マウスを用いて、ヒト喘息モデルである抗原誘発肺炎症モデルを作製し、アレルギー反応の解析を行った。結果は図５に示したとおりである。すなわち、動物にアレルゲンである卵白アルブミンと水酸化アルミニウムアジュバントの懸濁液を投与して感作した。感作から数週間後に、マウスにアレルゲンを投与して、アレルギー性の気道炎症反応を惹起した。ヘテロ欠損型は、アレルゲン投与後の好酸球の肺への浸潤が、野生型に比べて有意に軽微であることが観察された。ホモ欠損マウスでは、アレルゲン投与後の好酸球およびリンパ球の肺への浸潤が、野生型に比べて有意に軽微であることが観察された。
以上の結果から、この発明の遺伝子欠損マウスは、アレルギー発症の機構を解明するためのモデル動物として有用であることが確認された。
Example 2 (Measurement of allergic airway inflammation in gene-deficient mice)
Using the gene-deficient mouse obtained in Example 1, an antigen-induced lung inflammation model, which is a human asthma model, was prepared and allergic reaction was analyzed. The results are as shown in FIG. That is, the animals were sensitized by administering a suspension of ovalbumin as an allergen and an aluminum hydroxide adjuvant. Several weeks after sensitization, mice were administered allergens to elicit allergic airway inflammatory reactions. In the hetero-deficient type, it was observed that infiltration of eosinophils into the lung after allergen administration was significantly less than that in the wild type. In homo-deficient mice, the infiltration of eosinophils and lymphocytes into the lung after allergen administration was observed to be significantly less than in the wild type.
From the above results, it was confirmed that the gene-deficient mouse of the present invention is useful as a model animal for elucidating the mechanism of allergy development.
実施例３（遺伝子欠損マウスの急性気道炎症の測定）
実施例１で得た遺伝子欠損マウスを用いて、ヒト敗血症モデルであるリポポリサッカライド誘発肺炎症モデルを作製し、急性炎症反応の解析を行った。結果は図６に示したとおりである。すなわち、動物に１０μｇ／ｋｇのリポポリサッカライドを投与して、急性の気道炎症反応を惹起した。ホモ欠損マウスでは、リポポリサッカライド投与後の好中球の肺への浸潤が、野生型に比べて有意に増加していることが観察された。
以上の結果から、この発明の遺伝子欠損マウスは、急性炎症の発症機構を解明するためのモデル動物として有用であり、新規抗炎症物質をスクリーニングする系としても有効であることが確認された。
Example 3 (Measurement of acute airway inflammation in gene-deficient mice)
Using the gene-deficient mouse obtained in Example 1, a lipopolysaccharide-induced lung inflammation model, which is a human sepsis model, was prepared and analyzed for acute inflammatory reaction. The results are as shown in FIG. That is, 10 μg / kg lipopolysaccharide was administered to animals to elicit an acute airway inflammatory reaction. In homo-deficient mice, it was observed that neutrophil infiltration into the lung after lipopolysaccharide administration was significantly increased compared to wild-type mice.
From the above results, it was confirmed that the gene-deficient mouse of the present invention is useful as a model animal for elucidating the onset mechanism of acute inflammation, and is also effective as a system for screening a novel anti-inflammatory substance.
実施例４（遺伝子欠損マウスの組織傷害の測定）
実施例１で得た遺伝子欠損マウスに外科的に傷害を与え、損傷部位の治癒の程度を解析した。結果は図７に示したとおりである。すなわち、マウスの頭部に外科的傷害を与え、数日後の組織損傷の程度を組織化学的に調べたところ、ホモ欠損マウスは、野生型マウスに比べて治癒が早いことが確認された。
以上の結果から、この発明の遺伝子欠損マウスは、組織傷害の修復機構を解明するためのモデル動物として有用であり、新規傷創治療物質をスクリーニングする系としても有効であることが確認された。
Example 4 (Measurement of tissue injury in gene-deficient mice)
The gene-deficient mouse obtained in Example 1 was surgically damaged, and the degree of healing at the damaged site was analyzed. The results are as shown in FIG. That is, when a surgical injury was given to the head of the mouse and the degree of tissue damage after several days was examined histochemically, it was confirmed that the homo-deficient mouse was healed faster than the wild-type mouse.
From the above results, it was confirmed that the gene-deficient mouse of the present invention is useful as a model animal for elucidating the repair mechanism of tissue injury, and is also effective as a system for screening a novel wound wound treatment substance.
本発明は、医薬品の分野、特に、免疫系機能、中枢神経系機能、循環器系機能および生殖機能等に、先天性または反応性の障害を呈する遺伝子欠損マウスと、この遺伝子欠損マウスを用いて上記機能障害に対する予防または治療薬剤のスクリーニングの分野などにおいて利用可能である。 The present invention relates to a gene-deficient mouse that exhibits a congenital or reactive disorder in the field of pharmaceuticals, in particular, immune system function, central nervous system function, circulatory system function, and reproductive function, and the like. It can be used in the field of screening for preventive or therapeutic drugs for the above functional disorders.
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Non-Patent Citations (6)
| Title |
|---|
| JPN6012037234; Ｅｕｒ．Ｊ．Ｂｉｏｃｈｅｍ．， ２０００， Ｖｏｌ．２６７， ｐ．３３１５-３３２２ * |
| JPN6012037241; 日常生活における快適な睡眠の確保に関する総合研究 （第Ｉ期 平成８年〜１０年度）、２０００年３月、ｐ． * |
| JPN6012037244; Ｊ．Ｋｙｏｔｏ Ｐｒｅｆ． Ｕｎｉｖ． Ｍｅｄ．， １９８２， Ｖｏｌ．９１， Ｎｏ．８， ｐ．６６９-６７１ * |
| JPN6012037247; Ｊ．Ｃｅｌｌ．Ｐｙｓｉｏｌ．， １９８９， Ｖｏｌ．１４１， Ｎｏ．２， ｐ．２７５-２８０ * |
| JPN6012037250; Ｅｉｃｏｓａｎｏｉｄｓ， １９９０， Ｖｏｌ．３， Ｎｏ．４， ｐ．２１３-２１８ * |
| JPN6012037252; Ｂｉｏｃｈｅｍ．Ｂｉｏｐｈｙｓ．Ｒｅｓ．Ｃｏｍｍ．， １９９１， Ｖｏｌ．１７４， Ｎｏ．２， ｐ．７５８-７６６ * |
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