JP2006087384A - Animal exhibiting short or long period circadian rhythm - Google Patents
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Abstract
Description
本発明は睡眠異常のモデル動物に関する。 The present invention relates to a model animal having abnormal sleep.
本発明は、日周期リズム(circadian rhythm)の分子機構の解明を目的とした研究において有効なモデル動物、より詳しくは、体温、自発的活動量、覚醒時間および睡眠等の日周期リズムが正常動物より短周期または長周期を示す動物に関する。また本発明は、本発明の動物の作製方法、ならびに本発明の動物の日周期リズムに影響を及ぼす化合物のスクリーニング方法に関する。 The present invention relates to a model animal effective in research aimed at elucidating the molecular mechanism of circadian rhythm, and more specifically, an animal with normal circadian rhythm such as body temperature, spontaneous activity, awakening time, and sleep. It relates to animals that exhibit shorter or longer cycles. The present invention also relates to a method for producing the animal of the present invention and a method for screening a compound that affects the circadian rhythm of the animal of the present invention.
生物に内在する約２４時間の日周期リズムは、体内時計(または生物時計)と呼ばれる機構によって制御されていると考えられている。ヒトを対象とした臨床研究においては、睡眠相前進症候群や睡眠相後退症候群、不規則型睡眠覚醒障害、躁鬱病、あるいは身近なところでは時差ぼけ、シフトワークによる睡眠障害など、様々な疾病が体内時計の異常によって引き起こされていると考えられている。 The circadian rhythm of about 24 hours inherent in living organisms is thought to be controlled by a mechanism called a biological clock (or biological clock). In clinical research on human subjects, various diseases such as advanced sleep phase syndrome and late sleep phase syndrome, irregular sleep-wake disorder, manic depression, or jet lag in the immediate vicinity, sleep disorder due to shift work, etc. It is believed to be caused by a clock abnormality.
これまでに、哺乳類において生物時計を制御する遺伝子がいくつか同定されており、これには、Period（Per1,Per2,Per3）、Clock、BMAL、Cryptochrome（Cry１、Cry２）などが含まれる。Per遺伝子産物のPERは体内時計の中枢である視交叉上核でも各臓器でも１日周期の増減を示す分子であり、CLOCK/BMALによる転写活性化を抑制する因子といわれている。PER2のノックアウトマウスがリズム異常を示す一方で、PER1やPER3のノックアウトマウスでは顕著なリズム異常が見られないことから、３種類あるPERのなかでもPER2が生物時計を制御する上で最も重要な分子であると考えられている。 To date, several genes that control biological clocks in mammals have been identified, including Period (Per1, Per2, Per3), Clock, BMAL, Cryptochrome (Cry1, Cry2) and the like. The PER gene product, PER, is a molecule that exhibits an increase or decrease in the daily cycle in both the suprachiasmatic nucleus, which is the center of the body clock, and in each organ, and is said to be a factor that suppresses transcriptional activation by CLOCK / BMAL. While PER2 knockout mice show rhythm abnormalities, while PER1 and PER3 knockout mice do not show any significant rhythm abnormalities, among them, PER2 is the most important molecule for controlling biological clocks. It is considered to be.
日周期リズムに関わる体内時計の全体像、その詳しいメカニズム等の情報を提供することは、基礎研究はもとより、睡眠や恒常性の維持、投薬間隔や薬効の至適時間帯等をはじめとするヒトの生体リズムの関与する各種臨床的な観点からも極めて重要な課題となっている。 Providing information on the whole body of the circadian clock related to the circadian rhythm, its detailed mechanism, etc. is not only for basic research, but also for humans, including sleep and homeostasis, medication intervals, optimal time periods for drug efficacy, etc. It is an extremely important issue from various clinical viewpoints related to the biological rhythms.
したがって、睡眠異常などを引き起こす元となるリズム異常のメカニズムを解明し、睡眠異常を治療する方法を開発するために、種々の型の睡眠異常を示すモデル動物が必要とされている。 Therefore, in order to elucidate the mechanism of rhythm abnormalities that cause sleep abnormalities and to develop methods for treating sleep abnormalities, model animals that exhibit various types of sleep abnormalities are needed.
これまでに、時計遺伝子の変異と睡眠異常との関連性を示すいくつかの例が報告されている。本発明者らは、Clock遺伝子に変異が生じたマウスが日周期リズムに異常を示し、夜型睡眠モデルマウスとして有用であることを見いだした（特開２００３−７０７３７６）。Zhengらは、ＰＡＳ領域を欠失したmPer2遺伝子を有するマウスが、短周期型の日周期リズムを示すことを報告している（Nature 400, 169-173, 1999）。また、家族性睡眠症候群という疾患においてPER2分子の中央部分にあるカゼインキナーゼによるリン酸化部位に遺伝子多型(SNPS)が存在していて、それが原因で睡眠異常が生ずるケースがあるとの報告がある。しかし、安定的に短周期型または長周期型の日周期リズムを示すモデル動物は知られていない。
本発明は、ヒトにおける日周期リズム異常のメカニズムを解明するための手段として有効な、睡眠異常を示すモデル動物を提供することを目的とする。具体的には、本発明は日周期リズムが短周期型または長周期型を示すモデル動物を提供することを目的とする。 An object of the present invention is to provide a model animal showing sleep abnormalities that is effective as a means for elucidating the mechanism of abnormal circadian rhythms in humans. Specifically, an object of the present invention is to provide a model animal in which the daily rhythm is a short period type or a long period type.
本発明者らは、動物において野生型または変異型のPer2遺伝子を強制発現させることにより、その動物の日周期リズムが短周期型または長周期型となることを見いだした。すなわち本発明は、外来のPer2遺伝子を有する非ヒトトランスジェニック動物を提供する。本発明のトランスジェニック動物は、短周期型または長周期型の日周期リズムを示す。本発明の好ましい態様においては、本発明のトランスジェニック動物は、核移行配列が欠失したPer2変異型遺伝子を有し、長周期型の日周期リズムを示す。 The present inventors have found that the circadian rhythm of an animal becomes a short period type or a long period type by forcibly expressing the wild type or mutant Per2 gene in the animal. That is, the present invention provides a non-human transgenic animal having a foreign Per2 gene. The transgenic animal of the present invention exhibits a short-period or long-period circadian rhythm. In a preferred embodiment of the present invention, the transgenic animal of the present invention has a Per2 mutant gene lacking a nuclear translocation sequence and exhibits a long-period circadian rhythm.
別の観点においては、本発明は、上述の本発明のトランスジェニック動物を作製する方法であって、動物細胞中で機能するプロモーターの下流に動作可能なように連結されたPer2遺伝子を動物の胚細胞に導入し、前記胚細胞からトランスジェニック動物を発生させることを含む方法を提供する。 In another aspect, the present invention provides a method for producing the above-described transgenic animal of the present invention, wherein a Per2 gene operably linked downstream of a promoter that functions in an animal cell is transferred to an animal embryo. A method comprising introducing into a cell and generating a transgenic animal from said embryonic cell is provided.
また別の観点においては、本発明は、睡眠異常に影響を及ぼす化合物をスクリーニングする方法であって、試験化合物を上述の本発明のトランスジェニック動物に投与し、前記動物の日周期リズムを測定し、日周期リズムを変化させる化合物を同定する、の各工程を含む方法を提供する。 In another aspect, the present invention is a method for screening a compound that affects sleep abnormalities, comprising administering a test compound to the above-described transgenic animal of the present invention, and measuring the circadian rhythm of the animal. A method comprising the steps of: identifying a compound that alters the circadian rhythm.
本発明のトランスジェニック動物は、外来のPer2遺伝子を体細胞中に有することを特徴とする。「外来のPer2遺伝子」とは、トランスジェニック動物が元々その染色体上に持っているPer2遺伝子の代わりにまたはそれに加えて、外から導入されたPer2遺伝子を意味する。外来のPer2遺伝子は、ホモで存在していてもヘテロで存在していてもよい。外来のPer2遺伝子は、動物細胞中で機能するプロモーターの下流に動作可能なように連結して、トランスジェニック動物に導入する。好ましくは、プロモーターはPer2遺伝子を強制的に、すなわち、天然のPer2遺伝子の発現の制御とは独立して発現させる。プロモーターは構成的なものでも誘導可能なものでもよい。 The transgenic animal of the present invention is characterized by having a foreign Per2 gene in somatic cells. “Exogenous Per2 gene” means a Per2 gene introduced from outside instead of or in addition to the Per2 gene originally present on the chromosome of the transgenic animal. The exogenous Per2 gene may be present in homo or hetero. The exogenous Per2 gene is operably linked downstream of a promoter that functions in animal cells and introduced into the transgenic animal. Preferably, the promoter forces the Per2 gene to be expressed, i.e. independent of the control of the expression of the native Per2 gene. The promoter may be constitutive or inducible.
PER2（PERIOD2）は、哺乳動物の日周期を制御する蛋白質の１つである。しかし、PER2がどのようなメカニズムで日周期を制御しているのかは不明であった。本発明においては、野生型のPER2を強制発現させたトランスジェニックマウスが短周期型の日周期を示し、核移行配列を欠失したPER2を強制発現させたトランスジェニックマウスが長周期型の日周期を示すことが見いだされた。PER2の「核移行配列」とは、PER2の分子中に存在し、PER2蛋白質の核内への移行を指示するシグナルとなるアミノ酸配列である。本発明者らは、これまでに、培養細胞を用いた研究により、PER2の分子中に存在する核内移行配列は、通常はこの蛋白質の他の領域によりマスクされていること、PER2がCRYと結合すると核内移行配列がアクティブとなってPER2／CRY複合体が核内へと移行すること、PER2はＣ末端領域中にCRYと結合するための結合部位を有しており、この結合部位を欠失させるとPER2もCRYも核内へ移行できなくなること、ならびに、PER2の核移行配列を欠失させると、PER2だけでなくCRYも核内へ移行できなくなることを明らかにした（Mol. Cell. Biol. 21 (19), 6651-6659 (2001)）。ＣＲＹは核内で転写抑制因子として働くことが知られているため、変異型PER2の存在下では核内移行が阻害され、転写因子としての機能が果たせなくなると考えられる。 PER2 (PERIOD2) is one of the proteins that control the circadian cycle of mammals. However, it was unclear what mechanism PER2 controls the circadian cycle. In the present invention, transgenic mice in which wild-type PER2 is forcibly expressed show a short-period type circadian cycle, and transgenic mice in which PER2 forcibly expresses a nuclear translocation sequence is a long-period type circadian cycle Was found to show. The “nuclear translocation sequence” of PER2 is an amino acid sequence that is present in the PER2 molecule and serves as a signal that directs translocation of the PER2 protein into the nucleus. The present inventors have previously conducted studies using cultured cells that the nuclear translocation sequence present in the molecule of PER2 is usually masked by other regions of this protein, and that PER2 is CRY and CRY. When bound, the nuclear translocation sequence becomes active and the PER2 / CRY complex moves into the nucleus. PER2 has a binding site for binding to CRY in the C-terminal region. It has been clarified that deletion of PER2 and CRY cannot translocate into the nucleus when deleted, and that deletion of PER2 nuclear translocation sequence prevents not only PER2 but also CRY from translocating into the nucleus (Mol. Cell Biol. 21 (19), 6651-6659 (2001)). Since CRY is known to act as a transcriptional repressor in the nucleus, it is thought that in the presence of mutant PER2, nuclear translocation is inhibited and the function as a transcription factor cannot be performed.
下記の実施例に記載されるように、本発明のトランスジェニック動物は、日周期リズムが変化し、短周期型または長周期型の日周期リズムを示す。「日周期リズム」とは、ほぼ24時間周期で繰り返す生理現象を意味する。「短周期型」とは、対象とする動物を恒暗条件下で飼育し、特定の生理学的パラメーターにより日周期リズムを測定した場合に、活動期（覚醒期）と休止期が現れる周期が正常動物より短いことを意味する。「長周期型」とは、対象とする動物を恒暗条件下で飼育し、特定の生理学的パラメーターにより日周期リズムを測定した場合に、活動期（覚醒期）と休止期が現れる周期が正常動物より長いことを意味する。 As described in the Examples below, the transgenic animals of the present invention have a circadian rhythm that changes, exhibiting a short-period or long-period circadian rhythm. “Diurnal rhythm” means a physiological phenomenon that repeats in a cycle of approximately 24 hours. “Short cycle type” means that the period in which the active period (wakefulness period) and rest period appear when the target animal is raised under constant dark conditions and the circadian rhythm is measured by specific physiological parameters is normal. Means shorter than animals. “Long-period type” means that the period in which the active period (wakefulness period) and rest period appear is normal when the target animal is raised under constant dark conditions and the circadian rhythm is measured by specific physiological parameters. Means longer than animals.
日周期リズムを測定するための生理学的パラメーターとしては、限定するものではないが、例えば飲水行動、体温、自発的活動量および覚醒時間を挙げることができる。これらの生理学的パラメーターの日周期リズムは、以下に示すようにして測定することができる。 Examples of physiological parameters for measuring the circadian rhythm include, but are not limited to, drinking behavior, body temperature, spontaneous activity, and arousal time. The circadian rhythm of these physiological parameters can be measured as shown below.
飲水行動；給水瓶の吸水口前面部に赤外線センサーを装着し、動物が飲水した場合センサーが感知し、その活動を記録する。５分毎に活動の有無を決定し経時的な行動量を評価する。 Drinking behavior: An infrared sensor is attached to the front of the water inlet of the water bottle. When an animal drinks water, the sensor detects it and records its activity. The presence or absence of activity is determined every 5 minutes, and the amount of behavior over time is evaluated.
体温；動物の腹腔内に体温の電波遠隔測定装置（TA10TA-F20; Data SciencesInt., USA）を埋め込み、これを介して測定する。得られた測定値を1時間ごとに平均化する。 Body temperature: A radio telemetry device for body temperature (TA10TA-F20; Data Sciences Int., USA) is implanted in the abdominal cavity of an animal, and the measurement is performed therethrough. The obtained measurements are averaged every hour.
自発的活動量；動物の首の両側の筋肉に筋電図（EMG）測定のためのテフロン（登録商標）コートされたステンレスワイヤーを埋め込み、該動物の動きに伴うシグナル強度の変化をモニターすることによって測定する。得られた測定値を1時間ごとに平均化する。 Spontaneous activity; Implanting Teflon-coated stainless steel wire for electromyogram (EMG) measurement into the muscles on both sides of the animal's neck and monitoring changes in signal intensity as the animal moves Measure by. The obtained measurements are averaged every hour.
覚醒時間；動物の首の両側の筋肉に筋電図（EMG）測定のためのテフロン（登録商標）コートされたステンレスワイヤーを、また該動物の頭蓋骨中に脳波（EEG）測定のためのステンレス小型ねじ式電極を埋め込み、コンピューター画面上で５秒間ごとの脳波および筋電図のパターンを視覚的に同定することによって定量化する。得られた値を1時間ごとに平均化する。 Awakening time: Teflon-coated stainless steel wire for electromyogram (EMG) measurement on the muscles on both sides of the animal's neck, and stainless steel small for electroencephalogram (EEG) measurement in the animal's skull Quantify by implanting screw electrodes and visually identifying the electroencephalogram and electromyogram patterns every 5 seconds on a computer screen. The obtained values are averaged every hour.
上記特性を示す本発明の動物としては、ヒトを除く哺乳動物であれば特に限定されないが、例えばマウス、ラット、ウサギ、ブタ、ウシ、ハムスターを挙げることができる。 The animal of the present invention exhibiting the above characteristics is not particularly limited as long as it is a mammal other than a human, and examples thereof include mouse, rat, rabbit, pig, cow and hamster.
本発明の短周期型または長周期型モデル動物は、新しい睡眠薬のスクリーニング（短周期型または長周期型を正常型に戻す）、日周期リズム分子機構の研究（特にPER2分子がどのように哺乳類日周期リズム分子機構形成に関わるかという点を明らかにするための実験動物になりうる）、睡眠異常の発症メカニズムの研究、ならびに疾患の起こりやすい時刻の分子機構の研究（例えば、心筋梗塞は朝方に多い、突然死は夜方に多い等）等に有用である。 The short-cycle or long-cycle model animals of the present invention can be used for screening new sleeping pills (returning short-cycle or long-cycle types to normal), studying circadian rhythm molecular mechanisms (particularly how PER2 molecules are It may be an experimental animal to clarify whether it is involved in the formation of a periodic rhythm molecular mechanism), research on the onset mechanism of sleep abnormalities, and molecular mechanism of the time at which the disease is likely to occur (for example, myocardial infarction in the morning Many, sudden deaths are more common at night, etc.).
本発明の日周期リズムが短周期型または長周期型を示すトランスジェニック動物は、以下のようにして作製することができる。Per2遺伝子の配列は、Genbank Accession No. AB016532から入手することができる。ラットPER2の核移行配列は、アミノ酸残基７７８−７９４に位置すると考えられている（Mol. Cell. Biol. 21 (19), 6651-6659 (2001)）。それらの配列は、マウスPER2のアミノ酸残基７７８−７９４、ヒトPER2のアミノ酸残基７８９−８０６に相当する。この核移行配列を欠失したPER2をコードする遺伝子は、制限酵素による消化とライゲーションを適宜組み合わせることにより、あるいは、ＰＣＲ反応等の手法により核移行配列の上流のフラグメントと下流のフラグメントとを作製した後にこれらを連結させることにより、または当該技術分野において知られる他の適当な方法により、作製することができる。 The transgenic animal in which the circadian rhythm of the present invention shows a short period type or a long period type can be produced as follows. The sequence of Per2 gene can be obtained from Genbank Accession No. AB016532. The nuclear translocation sequence of rat PER2 is thought to be located at amino acid residues 778-794 (Mol. Cell. Biol. 21 (19), 6651-6659 (2001)). These sequences correspond to amino acid residues 778-794 of mouse PER2 and amino acid residues 789-806 of human PER2. The gene encoding PER2 lacking the nuclear translocation sequence was prepared by combining a restriction enzyme digestion and ligation as appropriate, or by using a PCR reaction or the like to produce an upstream fragment and a downstream fragment of the nuclear translocation sequence. They can be made later by concatenating them or by other suitable methods known in the art.
このようにして製造した野生型または変異型PER2遺伝子を動物細胞中で作用しうる適当な発現ベクター中のプロモーターの下流に挿入して、遺伝子導入用のコンストラクトを構築する。プロモーターとしては、当該技術分野において知られる動物細胞中で作用しうるいずれのプロモーターを用いてもよく、例えば、ＣＡＧプロモーター、ＣＭＶプロモーター、ＳＶ４０プロモーターなどを用いることができる。 A wild-type or mutant PER2 gene produced in this way is inserted downstream of a promoter in an appropriate expression vector that can act in animal cells to construct a gene introduction construct. As the promoter, any promoter that can act in animal cells known in the art may be used, and for example, CAG promoter, CMV promoter, SV40 promoter and the like can be used.
採卵用の雌動物に、牝馬血清性腺刺激ホルモンを腹腔内投与し、次にヒト絨毛性性腺刺激ホルモンを投与した後、雄と交尾させる。前核期受精卵を採取し、野生型または変異型PER2遺伝子を含むコンストラクトを前核にマイクロインジェクションする。受精卵をｍ−ＫＲＢ培地中で数時間から一晩インキュベートした後、偽妊雌の卵管に移植する。生まれた仔のそれぞれについて、目的とする遺伝子が組み込まれているか否かについてスクリーニングする。スクリーニングは、各個体の尾部等から少量の血液を採取し、ＰＣＲ、サザンブロッティング等の方法を用いてＤＮＡを分析することにより行うことができる。所望のトランスジンを有する個体を選択する。得られたトランスジェニック動物と、野生型動物とを交配させてＦ１を取得する。 A marine serum gonadotropin is intraperitoneally administered to a female animal for egg collection, followed by human chorionic gonadotropin and then mated with a male. Pronuclear fertilized eggs are collected and a construct containing a wild type or mutant PER2 gene is microinjected into the pronucleus. Fertilized eggs are incubated in m-KRB medium for several hours to overnight and then transplanted into the oviduct of pseudopregnant females. Each born pup is screened for the integration of the gene of interest. Screening can be performed by collecting a small amount of blood from the tail of each individual and analyzing the DNA using a method such as PCR or Southern blotting. Individuals with the desired transgin are selected. F1 is obtained by mating the resulting transgenic animal with a wild-type animal.
さらに別の観点においては、本発明は、睡眠異常に影響を及ぼす化合物をスクリーニングする方法を提供する。該方法は、試験化合物を本発明のトランスジェニック動物に投与し、前記動物の日周期リズムを測定し、日周期リズムを変化させる化合物を同定する、の各工程を含む。ここで、「睡眠異常」とは、体内時計の制御の異常により生ずる疾病または状態を表し、例えば、睡眠睡眠相前進症候群、睡眠相後退症候群、不規則型睡眠覚醒障害、時差ぼけ、躁鬱病、痴呆老人の夜間徘徊などが含まれる。本発明の方法により睡眠異常に影響を及ぼす化合物として同定された化合物は、日周期リズムや睡眠異常のメカニズム研究用の試薬として有用である。さらに、このような化合物は、日周期リズムに異常を示す動物のリズムを変更する薬剤、すなわち新規な睡眠薬の候補として有用であると考えられる。 In yet another aspect, the present invention provides a method of screening for compounds that affect sleep abnormalities. The method includes the steps of administering a test compound to the transgenic animal of the present invention, measuring the circadian rhythm of the animal, and identifying a compound that alters the circadian rhythm. Here, “sleep anomaly” refers to a disease or condition caused by an abnormality in the control of the biological clock, such as sleep sleep phase advance syndrome, sleep phase regression syndrome, irregular sleep-wake disorder, jet lag, manic depression, Includes night-time illnesses for elderly people with dementia. Compounds identified as compounds that affect sleep abnormalities by the method of the present invention are useful as reagents for studying the mechanisms of circadian rhythm and sleep abnormalities. Furthermore, such a compound is considered useful as a candidate for a drug that changes the rhythm of an animal exhibiting an abnormality in the circadian rhythm, that is, a novel hypnotic.
以下に、実施例により本発明をより詳細に説明するが、これらの記載は例示の目的にのみ提供されるものであり、本発明の範囲はこれらに限定されない。 The present invention will be described in more detail with reference to the following examples. However, these descriptions are provided for illustrative purposes only, and the scope of the present invention is not limited thereto.
ＣＡＧプロモーターの下流に核移行配列をコードするＤＮＡ配列（１６４２−２４９０）を欠失したラットPer2遺伝子を連結したＤＮＡを作製した（図１）。ここから切り出したDNA断片をC57BL6系統マウスのES細胞にマイクロインジェクションし、ICRマウスの子宮内に着床後出産させた。産子の尾からDNAを抽出し、PCR法によりPer2遺伝子を検出することにより、相同組み換えにより染色体にPer2を含むDNA断片が挿入されたマウスを同定した。その結果12ラインのトランスジェニックマウスを得た。さらに、これらのトランスジェニックマウスを正常型c57BL6系統と交配させることにより産子を得た。その結果、PER2変異型蛋白質を発現させる変異動物１４ラインのうち、PER2変異型蛋白質を過剰発現するマウスを７ライン同定した。 A DNA in which the rat Per2 gene lacking the DNA sequence encoding the nuclear translocation sequence (1642-2490) was linked downstream of the CAG promoter was prepared (FIG. 1). The DNA fragment excised from this was microinjected into ES cells of C57BL6 strain mice, and were delivered after implantation in the uterus of ICR mice. By extracting DNA from the tail of the litter and detecting the Per2 gene by the PCR method, mice in which a DNA fragment containing Per2 was inserted into the chromosome by homologous recombination were identified. As a result, 12 lines of transgenic mice were obtained. Furthermore, offspring were obtained by crossing these transgenic mice with the normal c57BL6 strain. As a result, 7 lines of mice overexpressing the PER2 mutant protein were identified among 14 lines of mutant animals expressing the PER2 mutant protein.
これらのトランスジェニックマウスについて、以下のようにして日周行動リズムを測定した。恒暗条件下での飲水行動を測定した。給水瓶の吸水口前面部に赤外線センサーを装着し、動物が飲水した場合センサーが感知し、その活動を記録する。５分毎に活動の有無を決定し、活動が観察された場合を黒、ない場合をしろで表し、１日の活動パターンを表示した（図２、３）。トランスジェニックマウスの活動を１段目に１日目と２日目、２段目に２日目と３日目のデータを順次示すダブルプロット法で表示すると、２４時間以上の周期では左に傾いた黒い帯がみられ、長周期で活動していた（図２、３）。 With respect to these transgenic mice, the daily behavior rhythm was measured as follows. Drinking behavior under constant dark conditions was measured. An infrared sensor is attached to the front of the water inlet of the water bottle, and when the animal drinks water, the sensor detects it and records its activity. The presence / absence of activity was determined every 5 minutes, and when activity was observed, it was shown as black, and when there was no activity, the daily activity pattern was displayed (FIGS. 2 and 3). When the activity of the transgenic mouse is displayed by the double plot method in which the data on the first day and the second day are displayed in the first row and the data on the second and third days are sequentially displayed in the second row, the activity is inclined to the left in a cycle of 24 hours or more. A dark black band was seen and it was active in a long cycle (Figs. 2 and 3).
その結果、核移行配列欠失型PER2トランスジェニックマウスのうち、恒暗条件において測定したリズムが長周期となるものが２ライン同定された。その１つである０３−１ラインは、正常マウス（２３．６時間）と比較して、長周期の行動パターンを示し、その平均周期は２５．２時間であった（図２）。また別の０７−３ラインも、正常マウスと比較して、長周期の行動パターンを示し、その平均周期は２４．８時間であった（図３）。すなわち、核移行配列を欠失したPER2を導入することにより、長周期型の日周期リズムを示す動物を得ることができた。 As a result, two lines were identified among the nuclear translocation sequence-deficient PER2 transgenic mice whose rhythm measured under constant dark conditions had a long period. One of them, the 03-1 line, showed a long-period behavior pattern compared to normal mice (23.6 hours), and the average period was 25.2 hours (FIG. 2). Another 07-3 line also showed a long-period behavior pattern compared to normal mice, with an average period of 24.8 hours (FIG. 3). That is, by introducing PER2 lacking the nuclear translocation sequence, an animal having a long-period circadian rhythm could be obtained.
実施例１と同様にして、野生型PER2の過剰発現マウスを作成した。ＣＡＧプロモーターの下流にPER2遺伝子を連結し、Ｃ５７ＢＬ／６マウスバックグラウンドのトランスジェニックマウス０６−１を作成した。この０６−１ラインマウスは、正常マウスと比較して短周期の行動パターンを示し、その平均周期は２３時間であった（図４）。 In the same manner as in Example 1, wild type PER2 overexpressing mice were prepared. The PER2 gene was ligated downstream of the CAG promoter to produce a transgenic mouse 06-1 with a C57BL / 6 mouse background. The 06-1 line mice showed a short-cycle behavior pattern compared to normal mice, and the average cycle was 23 hours (FIG. 4).
Claims (6)
A method for screening a compound that affects sleep abnormalities, comprising administering a test compound to the transgenic animal according to any one of claims 1 to 4, measuring the circadian rhythm of the animal, and determining the circadian rhythm. A method comprising the steps of identifying a compound to be altered.
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