JP2006081496A - Knockout nonhuman mammal in which secretion of neurotrophic factor is suppressed - Google Patents
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Abstract
Description
本発明は、神経栄養因子の分泌を抑制したノックアウト非ヒト哺乳動物及びその利用に関する。 The present invention relates to a knockout non-human mammal that suppresses the secretion of neurotrophic factors and uses thereof.
Levi-Montalciniらは、マウス肉腫から分泌され脊髄後根神経節の増大を起こす液性因子を精製、同定して神経成長因子(nerve growth factor:NGF)と命名した。その後、NGFが神経系の細胞増殖や分化の調節、そして神経細胞の生存、機能維持といった神経栄養作用をもつことが明らかとなり、神経栄養因子(neurotrophin:NT)と提唱されるようになった。続いて、ブタの脳より培養後根神経節細胞の生存を維持するBDNF(brain derived neurotrophic factor)が分離、同定された。そして、この両者の共通アミノ酸構造の部分をプローブとして第３番目の神経栄養因子の遺伝子がクローニングされ、NT-3と命名された。その後もNT-4、NT-5と一群のNGFファミリーに属する神経栄養因子が同定されている。 Levi-Montalcini and colleagues purified and identified a humoral factor secreted from mouse sarcoma that causes the dorsal root ganglion to increase and named it nerve growth factor (NGF). Later, it became clear that NGF has neurotrophic effects such as regulation of cell proliferation and differentiation of the nervous system, survival of neurons, and maintenance of functions, and it has been proposed as a neurotrophic factor (NT). Subsequently, BDNF (brain derived neurotrophic factor), which maintains the survival of cultured dorsal root ganglion cells, was isolated and identified from pig brain. The gene for the third neurotrophic factor was cloned using the common amino acid structure of both as a probe and named NT-3. Since then, neurotrophic factors belonging to NT-4, NT-5 and a group of NGF families have been identified.
BDNFとNT-3は小脳の発生に重要であることが知られており、BDNFやNT-3のノックアウト哺乳動物では小脳の特定の溝の消失、小脳顆粒細胞の細胞死の上昇や移動の遅れ、プルキンエ細胞の樹状突起の低形成が知られている（Bates, B., et al., Nature Neuroscience 1999, 2(2):115-117; 及び Schwartz, P.M., et al., Neuron 1999, 19(2): 269-281）。しかしながら、BDNFやNT-3のノックアウト哺乳動物は比較的短期間で死亡してしまうため、精神疾患モデルマウスとしての有用性は低かった。 BDNF and NT-3 are known to be important for cerebellar development. In BDNF and NT-3 knockout mammals, loss of specific cerebellar grooves, increased cell death of cerebellar granule cells and delayed migration Hypodenogenesis of Purkinje cells is known (Bates, B., et al., Nature Neuroscience 1999, 2 (2): 115-117; and Schwartz, PM, et al., Neuron 1999, 19 (2): 269-281). However, since BDNF and NT-3 knockout mammals die in a relatively short period of time, their usefulness as a mental disease model mouse was low.
本発明者らは以前に、小脳においてBDNFとNT-3の分泌に関与する遺伝子CAPS2(Calcium-activated protein for secretion 2)を同定、報告した（Sadakata, T., et al., J. Neuroscience 2004, 24(1): 43-52）。この遺伝子はBDNFやNT-3を含む小胞が細胞膜と融合し、BDNFやNT-3が細胞外に放出される過程に関わることが示唆されている。 The present inventors have previously identified and reported a CAPS2 (Calcium-activated protein for secretion 2) gene involved in the secretion of BDNF and NT-3 in the cerebellum (Sadakata, T., et al., J. Neuroscience 2004). , 24 (1): 43-52). This gene has been implicated in the process by which vesicles containing BDNF and NT-3 fuse with the cell membrane and BDNF and NT-3 are released outside the cell.
本発明は、神経栄養因子分泌を抑制したノックアウト非ヒト哺乳動物を提供することを解決すべき課題とした。特に本発明は、長期間生存することができて精神疾患モデル動物として有用性の高い、マウス神経栄養因子の分泌を抑制したノックアウト非ヒト哺乳動物を提供することを解決すべき課題とした。 An object of the present invention is to provide a knockout non-human mammal with suppressed neurotrophic factor secretion. In particular, an object of the present invention is to provide a knockout non-human mammal that is capable of surviving for a long time and is highly useful as a model animal for mental illness and that suppresses the secretion of mouse neurotrophic factor.
本発明者らは、小脳においてBDNFとNT-3の分泌に関与する遺伝子CAPS2(Calcium-activated protein for secretion 2)を欠損させることにより、これらの神経栄養因子が細胞外に放出されず、小脳の発生が損なわれることを見出し、本発明を完成するに至った。 By deleting the gene CAPS2 (Calcium-activated protein for secretion 2) involved in the secretion of BDNF and NT-3 in the cerebellum, the present inventors do not release these neurotrophic factors to the outside of the cell, The inventors have found that generation is impaired, and have completed the present invention.
即ち、本発明によれば、CAPS2(Calcium-activated protein for secretion 2)遺伝子の発現が人為的に抑制されていることを特徴とするノックアウト非ヒト哺乳動物またはその一部が提供される。 That is, according to the present invention, there is provided a knockout non-human mammal or a part thereof, wherein the expression of CAPS2 (Calcium-activated protein for secretion 2) gene is artificially suppressed.
好ましくは、CAPS2(Calcium-activated protein for secretion 2)遺伝子の小脳での発現が人為的に抑制されているノックアウト非ヒト哺乳動物またはその一部が提供される。 Preferably, a knockout non-human mammal or a part thereof in which expression of CAPS2 (Calcium-activated protein for secretion 2) gene in the cerebellum is artificially suppressed is provided.
好ましくは、本発明のノックアウト非ヒト哺乳動物は、以下の所見のうち少なくとも1つ以上を示す。
（１）小脳のＶＩ葉とＶＩＩ葉の間の溝が欠失している。
（２）小脳における顆粒細胞の移動が遅滞しており、外顆粒層における細胞数が多い。
（３）小脳プルキンエ細胞の樹状突起の低形成が見られる。
Preferably, the knockout non-human mammal of the present invention exhibits at least one of the following findings.
(1) The groove between the cerebellar VI and VII leaves is missing.
(2) The migration of granule cells in the cerebellum is delayed and the number of cells in the outer granule layer is large.
(3) Low formation of dendrites of cerebellar Purkinje cells is observed.
好ましくは、本発明のノックアウト非ヒト哺乳動物は、CAPS2遺伝子の両方の対立遺伝子をともに変異遺伝子で置き換えたホモ接合体である。
好ましくは、本発明のノックアウト非ヒト哺乳動物は、CAPS2遺伝子の両方の対立遺伝子の1st exonをloxP遺伝子で挟んだPgk-neo遺伝子に置き換えたホモ接合体である。
Preferably, the knockout non-human mammal of the present invention is a homozygote in which both alleles of the CAPS2 gene are replaced with mutant genes.
Preferably, the knockout non-human mammal of the present invention is a homozygote in which the 1st exon of both alleles of the CAPS2 gene is replaced with a Pgk-neo gene sandwiched between loxP genes.
好ましくは、本発明のノックアウト非ヒト哺乳動物は、げっ歯類動物であり、特に好ましくはマウスである。 Preferably, the knockout non-human mammal of the present invention is a rodent, particularly preferably a mouse.
本発明の別の側面によれば、上記した本発明のノックアウト非ヒト哺乳動物またはその一部を神経細胞の異常に関連する疾患のモデル動物として使用する方法が提供される。
本発明のさらに別の側面によれば、上記した本発明のノックアウト非ヒト哺乳動物またはその一部を用いる、神経細胞の異常に関連する疾患の治療・予防薬のスクリーニング方法が提供される。
According to another aspect of the present invention, there is provided a method of using the above-described knockout non-human mammal of the present invention or a part thereof as a model animal for a disease associated with an abnormality of a nerve cell.
According to still another aspect of the present invention, there is provided a screening method for a therapeutic / prophylactic agent for a disease associated with nerve cell abnormality using the knockout non-human mammal of the present invention or a part thereof.
BDNFは様々な精神疾患に関与することが知られているが、BDNFノックアウトマウスは生後２日以内にその殆どが死亡してしまうために、精神疾患モデルマウスとしての有用性は低かった。本発明のCAPS2ノックアウトマウスは生後すぐに死亡するようなことはなく、ホモ接合体同士での交配も可能であるため、精神疾患に関するドラッグスクリーニングなどに利用可能である。また、小脳以外においてもCAPS2タンパク質は、(精神疾患で大いに注目されている)大脳皮質や黒質などでのBDNFの分泌に関与している可能性がその分布から示唆されているため、神経細胞の異常に関連する疾患（例えば、精神疾患など）のモデル動物としての有用性は高いと考えられる。 BDNF is known to be involved in various mental illnesses, but most of the BDNF knockout mice die within 2 days of life, and thus their usefulness as psychiatric disease model mice was low. Since the CAPS2 knockout mouse of the present invention does not die immediately after birth and can be crossed with homozygotes, it can be used for drug screening concerning psychiatric disorders. In addition to the cerebellum, the CAPS2 protein is likely to be involved in the secretion of BDNF in the cerebral cortex and substantia nigra (which has received much attention in psychiatric disorders). It is considered that the usefulness as a model animal of a disease (for example, a mental illness) related to abnormalities of the above is considered.
以下、本発明の実施の形態について詳細に説明する。
（Ａ）本発明のノックアウト非ヒト哺乳動物の特徴
本発明者らは今回、CAPS2遺伝子のノックアウト非ヒト哺乳動物を作製することに成功した。即ち、本発明のノックアウト非ヒト哺乳動物は、ゲノム中のCAPS2(Calcium-activated protein for secretion 2)遺伝子のＤＮＡ領域の一部を改変することにより、組織特異的に神経栄養因子の分泌を抑制したことを特徴とするものである。
Hereinafter, embodiments of the present invention will be described in detail.
(A) Characteristics of Knockout Non-Human Mammal of the Present Invention The present inventors have succeeded in producing a knockout non-human mammal of CAPS2 gene. That is, the knockout non-human mammal of the present invention suppressed the secretion of neurotrophic factor in a tissue-specific manner by modifying a part of the DNA region of CAPS2 (Calcium-activated protein for secretion 2) gene in the genome. It is characterized by this.
本発明の代表的なノックアウト非ヒト哺乳動物においては、CAPS2遺伝子の発現量の低下により、以下のような所見が認められる。
（１）小脳のＶＩ葉とＶＩＩ葉の間の溝が欠失している。
（２）小脳における顆粒細胞の移動が遅滞しており、外顆粒層における細胞数が多い。
（３）小脳プルキンエ細胞の樹状突起の低形成が見られる。
In the representative knockout non-human mammal of the present invention, the following findings are observed due to a decrease in the expression level of the CAPS2 gene.
(1) The groove between the cerebellar VI and VII leaves is missing.
(2) The migration of granule cells in the cerebellum is delayed and the number of cells in the outer granule layer is large.
(3) Low formation of dendrites of cerebellar Purkinje cells is observed.
非ヒト哺乳動物としては、特に限定されるものではなく、例えば、マウス、ハムスター、モルモット、ラット、ウサギ等のげっ歯類の他、ニワトリ、イヌ、ネコ、ヤギ、ヒツジ、ウシ、ブタ、サル等を使用することができる。これらのうち、実験動物として用いるには、作製、育成及び使用の簡便さなどの観点から見て、マウス、ハムスター、モルモット、ラット、ウサギ等のげっ歯類が好ましく、そのなかでも近交系が多数作出されており、受精卵の培養、体外受精等の技術が整っているマウスが特に好ましい。 The non-human mammal is not particularly limited. For example, in addition to rodents such as mice, hamsters, guinea pigs, rats, rabbits, chickens, dogs, cats, goats, sheep, cows, pigs, monkeys, etc. Can be used. Of these, rodents such as mice, hamsters, guinea pigs, rats, rabbits and the like are preferred for use as experimental animals from the viewpoint of ease of production, breeding and use, and among these, inbred strains are preferred. Mice that are produced in large numbers and are well-equipped with techniques such as culturing fertilized eggs and in vitro fertilization are particularly preferred.
（Ｂ）本発明のノックアウト非ヒト哺乳動物の作製
本発明のCAPS2遺伝子の発現が低下したノックアウト非ヒト哺乳動物は、公知の遺伝子組み換え法（ジーンターゲッティング法）により作製することができる。ジーンターゲッティング法は、当業者に公知の技術であり、各種実験書に従って行うことができる（例えば、村松正實、山本雅編集、『実験医学別冊 新訂 遺伝子工学ハンドブック 改訂第３版』（１９９９年、羊土社発行）などを参照）。
(B) knockout non-human mammal in which the expression of CAPS2 gene with reduced fabrication invention knockout non-human mammal of the present invention can be produced by known gene recombination method (gene targeting method). The gene targeting method is a technique known to those skilled in the art, and can be performed according to various experimental documents (for example, Masaaki Muramatsu, edited by Masaru Yamamoto, “Experimental Medicine Separate Volume, New Revised Genetic Engineering Handbook 3rd Edition” (1999, (Issued by Yodosha)).
先ず、CAPS2遺伝子のゲノムＤＮＡ断片を単離して、制限酵素地図を作製する。そして、その制限酵素地図に基づいて、CAPS2遺伝子の機能を低下させた配列を有するターゲティングベクターを下記の方法により作製することができる。具体的には、非ヒト哺乳動物のCAPS2遺伝子を含むＤＮＡを単離し、このＤＮＡ断片に適当な外来遺伝子（例えば、マーカー遺伝子など）と、エキソンの一部を切り出せるような位置にloxP配列とを挿入することによって、ターゲッティングベクターを構築することができる。 First, a genomic DNA fragment of the CAPS2 gene is isolated and a restriction enzyme map is prepared. Based on the restriction enzyme map, a targeting vector having a sequence with reduced CAPS2 gene function can be produced by the following method. Specifically, a DNA containing the CAPS2 gene of a non-human mammal is isolated, and an appropriate foreign gene (eg, a marker gene) is extracted from this DNA fragment, and a loxP sequence is located at a position where a part of the exon can be cut out. A targeting vector can be constructed by inserting.
ＤＮＡ断片に挿入する外来遺伝子としては、マーカー遺伝子が好ましく、特に、ネオマイシン耐性遺伝子などの抗生物質耐性遺伝子が好ましい。抗生物質耐性遺伝子を挿入した場合には、抗生物質を含む培地で培養するだけで相同組み換えを生じた細胞株を選択することができる。また、より効率的な選択を行うためにはターゲッティングベクターにチミジンキナーゼ遺伝子などを結合させておくこともできる。これにより、非相同組み換えを起こした細胞株を排除することができる。 As the foreign gene to be inserted into the DNA fragment, a marker gene is preferable, and an antibiotic resistance gene such as a neomycin resistance gene is particularly preferable. When an antibiotic resistance gene is inserted, a cell line that has undergone homologous recombination can be selected simply by culturing in a medium containing the antibiotic. In order to perform more efficient selection, a thymidine kinase gene or the like can be bound to a targeting vector. Thereby, the cell line which caused non-homologous recombination can be excluded.
loxP配列は、エキソンの一部を切り出せるような位置に挿入することが好ましい。例えば、CAPS2遺伝子の1st exonをloxP遺伝子で挟んだPgk-neo遺伝子で置き換えることができるようなターゲティングベクターを設計することが好ましい。 The loxP sequence is preferably inserted at a position where a part of the exon can be cut out. For example, it is preferable to design a targeting vector that can replace the 1 st exon of the CAPS2 gene with a Pgk-neo gene sandwiched between loxP genes.
本発明においては、哺乳動物の細胞にターゲッティングベクターを導入して、染色体上の目的遺伝子と相同組み換えを起こすことにより、目的のCAPS2遺伝子を破壊することができる。細胞にターゲッティングベクターを導入して、動物の個体又は子孫にその遺伝子を発現させる手法としては、（１）ＤＮＡを受精卵の前核期胚に注入する、（２）組換えレトロウイルスを初期胚に感染させる、（３）ターゲッティングベクターによる相同組換えを起こさせた胚性幹細胞（ＥＳ細胞）を胚盤胞又は８細胞期胚に注入する等の方法によって得られた宿主胚を動物 に移植して産仔を得、これを他の個体と交配する方法が挙げられる。目的とする遺伝子の相同組み換えが起こる頻度は低いことが知られており、目的とする相同組み換え体を得るためには、多数の組み換え体をスクリーニングする必要がある。しかし、受精卵では多数のスクリーニングを行うことが技術的に困難である。よって、受精卵と同様に多分化能を有し、かつｉｎ ｖｉｔｒｏで培養することができる細胞を使用することが好ましく、胚性幹細胞（ＥＳ細胞）を用いる上記（３）の方法が好ましい。現在、マウス由来のＥＳ細胞株としては複数のものが確立されており、下記の実施例で使用しているＥＳ細胞(MS12株)もその一つである。しかし、本発明で使用するＥＳ細胞はそれに限定されるものではなく、他の細胞株を使用することもできる。 In the present invention, the target CAPS2 gene can be destroyed by introducing a targeting vector into mammalian cells and causing homologous recombination with the target gene on the chromosome. Methods for introducing a targeting vector into a cell and expressing the gene in an animal individual or offspring include (1) injecting DNA into the pronuclear embryo of a fertilized egg, and (2) using a recombinant retrovirus as an early embryo. (3) A host embryo obtained by a method such as injecting embryonic stem cells (ES cells) that have undergone homologous recombination with a targeting vector into a blastocyst or an 8-cell stage embryo is transplanted into an animal. A method of obtaining a litter and mating it with another individual can be mentioned. It is known that the frequency of homologous recombination of the target gene is low, and in order to obtain the target homologous recombinant, it is necessary to screen a large number of recombinants. However, it is technically difficult to screen a large number of fertilized eggs. Therefore, it is preferable to use cells that have pluripotency and can be cultured in vitro like fertilized eggs, and the method of (3) above using embryonic stem cells (ES cells) is preferable. Currently, a plurality of mouse-derived ES cell lines have been established, and one of them is the ES cell (MS12 line) used in the following examples. However, the ES cells used in the present invention are not limited thereto, and other cell lines can be used.
本発明では、上記で得られたターゲティングベクター（相同組換え用ＤＮＡ）を、ＥＳ細胞に導入し、ＥＳ細胞中のCAPS2遺伝子との間で相同組換えを行うことが好ましい。ターゲティングベクターをＥＳ細胞に導入する方法としては、公知のエレクトロポレーション法、リポソーム法、リン酸カルシウム法、ＤＥＡＥ−デキストラン法等も利用できるが、導入遺伝子の相同組み換え効率を考えると、エレクトロポレーション法が好ましい。 In the present invention, it is preferable to introduce the targeting vector (DNA for homologous recombination) obtained above into ES cells and perform homologous recombination with the CAPS2 gene in the ES cells. As a method for introducing a targeting vector into an ES cell, a known electroporation method, liposome method, calcium phosphate method, DEAE-dextran method and the like can be used, but considering the homologous recombination efficiency of the transgene, the electroporation method is preferable.
次いで、得られた組み換えＥＳ細胞につき、相同組み換えが起こっているかどうかのスクリーニングを行う。例えば、ネオマイシン等の導入した薬剤耐性因子によりスクリーニングを行うことができる。更にスクリーニングを確実にするためには、サザンハイブリダイゼーション法やＰＣＲ法により、スクリーニングを行ってもよい。これらのアッセイにより、染色体上に存在する野生型CAPS2遺伝子と、導入したCAPS2遺伝子との間で正しく相同遺伝子組み換えが起った細胞を選択することができる。 Next, the obtained recombinant ES cells are screened for homologous recombination. For example, screening can be performed using a drug resistance factor such as neomycin. Furthermore, in order to ensure screening, screening may be performed by Southern hybridization or PCR. By these assays, cells in which homologous recombination has occurred correctly between the wild-type CAPS2 gene present on the chromosome and the introduced CAPS2 gene can be selected.
こうして得た変異CAPS2遺伝子を持つＥＳ細胞を、野生型動物の胚盤胞または８細胞期の胚内に導入する。そして、このＥＳ細胞胚を偽妊娠状態の仮親個体の子宮に移植し、出産させることによりキメラ動物を作製することができる。以下、マウスを例に挙げて説明する。 ES cells having the mutant CAPS2 gene thus obtained are introduced into blastocysts of wild-type animals or 8-cell stage embryos. A chimeric animal can be produced by transplanting this ES cell embryo into the uterus of a pseudo-parental temporary parent and giving birth. Hereinafter, a mouse will be described as an example.
ＥＳ細胞を胚盤胞等の初期胚に導入する方法としては、マイクロインジュクション法や凝集法が知られている。本発明では上記のいずれの方法を用いることも可能であり、当業者であれば適宜実施することができる。マウスの場合には、ホルモン剤（例えば、ＦＳＨ様作用を有するＰＭＳＧおよびＬＨ作用を有するｈＣＧを使用）により過排卵処理を施した雌マウスを、雄マウスと交配させることが好ましい。その後、胚盤胞を用いる場合には受精から３．５日目に、８細胞期胚を用いる場合には２．５日目に、それぞれ子宮から初期発生胚を回収する。このようにして回収した胚に対して、ターゲティングベクターを用いて相同組み換えを行ったＥＳ細胞をｉｎ ｖｉｔｒｏにおいて注入し、キメラ胚を作製することができる。 As a method for introducing ES cells into an early embryo such as a blastocyst, a microinjection method and an aggregation method are known. In the present invention, any of the above-described methods can be used, and those skilled in the art can appropriately implement them. In the case of mice, it is preferable to mate female mice that have undergone superovulation treatment with hormone agents (for example, using PMSG having FSH-like action and hCG having LH action) to male mice. Thereafter, early embryos are collected from the uterus on day 3.5 after fertilization when using blastocysts and on day 2.5 when using 8-cell stage embryos. The embryos thus recovered can be injected in vitro with ES cells that have undergone homologous recombination using a targeting vector to produce a chimeric embryo.
一方、仮親とするための偽妊娠雌マウスは、正常性周期の雌マウスを、精管結紮などにより去勢した雄マウスと交配することにより得ることができる。作出した偽妊娠マウスに対して、上記の方法により作製したキメラ胚を子宮内移植し、妊娠・出産させることによりキメラマウスを作製することができる。キメラ胚の着床、妊娠がより確実に起こるようにするため、受精卵を採取する雌マウスと仮親となる偽妊娠マウスとを、同一の性周期にある雌マウス群から作出することが望ましい。 On the other hand, a pseudopregnant female mouse for use as a foster parent can be obtained by mating a female mouse having a normal cycle with a male mouse castrated by vagina ligation or the like. A chimeric mouse can be produced by transplanting the chimeric embryo produced by the above method into the uterus to the produced pseudopregnant mouse and allowing it to become pregnant / deliver. In order to ensure that implantation and pregnancy of the chimeric embryo occur more reliably, it is desirable to produce a female mouse from which a fertilized egg is collected and a pseudopregnant mouse as a foster parent from a group of female mice in the same sexual cycle.
ＥＳ細胞移植胚に由来するマウス個体が、このようなキメラマウスの中から得られた場合、このキメラマウスを純系のマウスと交配し、そして次世代個体にＥＳ細胞由来の被毛色が現れることにより、ＥＳ細胞がキメラマウス生殖系列へ導入されたことを確認することができる。ＥＳ細胞が生殖系列へ導入されたことを確認するには、様々な形質を指標として用いることができるが、確認の容易さを考慮して、被毛色によることが望ましい。マウスにおいては野ネズミ色（アグーチ色）、黒色、黄土色、チョコレート色および白色などの被毛色が知られているが、使用するＥＳ細胞の由来系統を考慮して、キメラマウスと交配させるマウス系統を適宜選択することができる。また、体の一部（例えば尾部先端）からＤＮＡを抽出し、サザンブロット解析やＰＣＲアッセイを行うことにより、選抜を行うことも可能である。 When an individual mouse derived from an ES cell-transplanted embryo is obtained from such a chimeric mouse, this chimeric mouse is crossed with a pure mouse, and the ES cell-derived hair color appears in the next generation individual. It can be confirmed that ES cells have been introduced into the germline of chimeric mice. In order to confirm that the ES cell has been introduced into the germ line, various traits can be used as an index. However, in consideration of ease of confirmation, it is desirable to use a coat color. Although mouse hair colors such as wild mouse color (Agouti color), black color, ocher color, chocolate color and white color are known, mouse strains to be mated with chimeric mice in consideration of the ES cell origin line used Can be appropriately selected. It is also possible to select by extracting DNA from a part of the body (for example, the tip of the tail) and performing Southern blot analysis or PCR assay.
このキメラマウスを適当な系統の野生型個体と交配することによりＦ１ヘテロ型の産仔を得ることができる。キメラマウスの生殖細胞が相同組換え体、すなわちCAPS2遺伝子に外来遺伝子が挿入されているＥＳ細胞に由来していれば、CAPS2遺伝子の片方の対立遺伝子に外来遺伝子が導入されている個体（ヘテロ接合体）を得ることができる。さらに、このヘテロ接合体マウス同士を交配することにより、変異caps2遺伝子のホモ接合体のノックアウトマウスを作出することができる。 F1 heterozygous offspring can be obtained by crossing this chimeric mouse with a wild-type individual of an appropriate strain. If the germ cell of a chimeric mouse is derived from a homologous recombinant, ie, an ES cell in which a foreign gene is inserted into the CAPS2 gene, an individual in which the foreign gene is introduced into one allele of the CAPS2 gene (heterozygous) Body). Further, by crossing these heterozygous mice, a homozygous knockout mouse of the mutant caps2 gene can be produced.
上記方法により作出された本発明のノックアウト非ヒト哺乳動物は、雄性・雌性の組み合わせとして取得しておけば、それ以降は、必要に応じて適宜繁殖させることにより、容易に同じ遺伝子型のノックアウト動物を必要なだけ取得することができる。 If the knockout non-human mammal of the present invention produced by the above method is obtained as a male / female combination, then the knockout animal of the same genotype can be easily produced by appropriately breeding as necessary. You can get as many as you need.
また、上記した本発明のノックアウト非ヒト哺乳動物の一部としては、非ヒト哺乳動物の細胞、細胞内小器官、組織および臓器のほか、頭部、指、手、足、腹部、尾などが挙げられ、これらも全て本発明の範囲内に属する。また、本発明のノックアウト非ヒト哺乳動物から得られた細胞（特に、精子、卵細胞、ＥＳ細胞、体性幹細胞）は、本発明のノックアウト非ヒト哺乳動物の作出や、他の遺伝子を破壊したダブルノックアウト動物の作出に有用である。 Examples of the knockout non-human mammal of the present invention include non-human mammal cells, intracellular organelles, tissues and organs, as well as the head, fingers, hands, feet, abdomen, tail and the like. All of which are within the scope of the present invention. In addition, cells obtained from the knockout non-human mammal of the present invention (especially, sperm, egg cells, ES cells, and somatic stem cells) can be produced from the generation of the knockout non-human mammal of the present invention or double-disrupted other genes. Useful for creating knockout animals.
（Ｃ）本発明のノックアウト非ヒト哺乳動物の利用（有用性）
本発明のノックアウト非ヒト哺乳動物は、CAPS2遺伝子の発現が人為的に抑制されているものであり、CAPS2遺伝子の機能解析に利用することができる。また、CAPS2遺伝子は小脳においてBDNFとNT-3の分泌に関与することから、本発明のノックアウト非ヒト哺乳動物は、小脳におけるBDNFやNT-3の生理機能、特に、BDNFレベルの低下やNT-3レベルの低下に起因する様々な神経系発達・機能障害の分子機構の解明に有用である。即ち、本発明のノックアウト非ヒト哺乳動物は、神経細胞の異常に関連する疾患の病態解析やその診断・治療法の開発に極めて有用なモデル動物となりうる。
(C) Use (usefulness) of the knockout non-human mammal of the present invention
The knockout non-human mammal of the present invention has an artificially suppressed expression of the CAPS2 gene and can be used for functional analysis of the CAPS2 gene. In addition, since the CAPS2 gene is involved in the secretion of BDNF and NT-3 in the cerebellum, the knockout non-human mammal of the present invention has a physiological function of BDNF and NT-3 in the cerebellum, in particular, a decrease in BDNF levels and NT- It is useful for elucidating the molecular mechanisms of various nervous system development and dysfunctions caused by three levels of decrease. That is, the knockout non-human mammal of the present invention can be an extremely useful model animal for pathological analysis of diseases related to abnormalities of nerve cells and development of diagnosis and treatment methods.
さらに、本発明のノックアウト非ヒト哺乳動物は、神経細胞の異常に関連する疾患の治療・予防薬のスクリーニング、薬理試験、安全性試験などにも有用である。スクリーニング方法としては、ノックアウト哺乳動物を用いた公知の方法、例えば、被験物質を本発明のノックアウト非ヒト哺乳動物に静注または経口投与等により投与し、その治療効果を個体、器官、組織、細胞の各レベルで観察・確認したり、CAPS2遺伝子の小脳における発現量や小脳におけるBDNFとNT-3の含量を測定する方法を挙げることができる。 Furthermore, the knockout non-human mammal of the present invention is also useful for screening for therapeutic / prophylactic drugs for diseases associated with neuronal abnormalities, pharmacological tests, safety tests, and the like. As a screening method, a known method using a knockout mammal, for example, a test substance is administered to the knockout non-human mammal of the present invention by intravenous injection or oral administration, and the therapeutic effect thereof is obtained in individuals, organs, tissues, cells. Can be observed and confirmed at each level, or the expression level of CAPS2 gene in the cerebellum and the content of BDNF and NT-3 in the cerebellum can be exemplified.
また、本発明のノックアウト非ヒト哺乳動物に被験物質を投与し、投与後におけるCAPS2遺伝子の発現状態、又は小脳などにおけるBDNFやNT-3の含量を分析することにより、BDNFやNT-3の生理機能、特にBDNFレベルやNT-3レベルの低下に起因する様々な神経系発達・機能障害に対する治療効果や予防効果を評価することもできる。 Further, by administering a test substance to the knockout non-human mammal of the present invention and analyzing the expression state of the CAPS2 gene after administration, or the content of BDNF and NT-3 in the cerebellum, etc., the physiology of BDNF and NT-3 It is also possible to evaluate the therapeutic effects and preventive effects on various nervous system development / function disorders caused by a decrease in function, particularly BDNF level and NT-3 level.
すなわち、本発明のノックアウト非ヒト哺乳動物を利用することにより、BDNFレベルやNT-3レベルの低下に起因する様々な神経系発達・機能障害の治療効果及び予防効果を評価することが可能であり、本発明のノックアウト非ヒト哺乳動物は、上記障害の病態評価モデル動物として利用することができる。例えば、本発明のノックアウト非ヒト哺乳動物を用いて、これらの病態回復および重篤程度を判定し、この疾患の治療方法の検討を行うことも可能である。
以下の実施例により本発明をさらに具体的に説明するが、本発明は実施例によって限定されるものではない。
That is, by using the knockout non-human mammal of the present invention, it is possible to evaluate the therapeutic effects and preventive effects of various nervous system development / function disorders caused by the decrease in BDNF levels and NT-3 levels. The knockout non-human mammal of the present invention can be used as a disease state evaluation model animal for the above-mentioned disorder. For example, by using the knockout non-human mammal of the present invention, it is possible to determine the recovery and severity of these pathological conditions and to examine a method for treating this disease.
The following examples further illustrate the present invention, but the present invention is not limited to the examples.
実施例１：CAPS2遺伝子のノックアウトマウスの作製
（１）ターゲッティングベクターの作製
マウスのCAPS2遺伝子の1st exonをloxP遺伝子で挟んだPgk-neo遺伝子に置き換えるためのターゲッティングベクターを作製した。図１に示したように、caps2遺伝子の1st exon上流のXho I-SmaI断片と1st exon下流のSmaI-EcoR I断片の間に、loxP遺伝子で挟んだPgk-neo遺伝子を逆向きに挿入した。また図１に示したように、ネガティブ選択としてpMClDT-A (constructed by Takeshi Yagi)でdriveされるdiphtheria toxin A-fragment遺伝子カセットを５’側に挿入した。相同組換えが起こった場合は最下段に示したような置き換えが起こるため、マウスゲノムをBamHIまたはEcoR Vで切断後、Southern blottingをそれぞれ図１に記載の「プローブ(1)」または「プローブ(2)」で行うことにより、相同組換えが起こったクローンについてはバンドシフトとして検出される。
Example 1: Preparation of knockout mouse of CAPS2 gene (1) Preparation of targeting vector A targeting vector for replacing the 1st exon of the mouse CAPS2 gene with the Pgk-neo gene sandwiched between loxP genes was prepared. As shown in FIG. 1, a Pgk-neo gene sandwiched between loxP genes was inserted in the reverse direction between the Xho I-SmaI fragment upstream of 1st exon and the SmaI-EcoR I fragment downstream of 1st exon of the caps2 gene. Further, as shown in FIG. 1, a diphtheria toxin A-fragment gene cassette driven by pMClDT-A (constructed by Takeshi Yagi) was inserted into the 5 ′ side as a negative selection. When homologous recombination occurs, replacement as shown in the bottom row occurs. Therefore, after cutting the mouse genome with BamHI or EcoR V, Southern blotting is performed using the “probe (1)” or “probe ( By performing the procedure in 2), a clone in which homologous recombination has occurred is detected as a band shift.
（２）ＥＳ細胞の培養
ＥＳ細胞(MS12株)、ＥＦ細胞の培養には次に記す培地を使用する。
ＥＳ細胞(MS12株)の由来は、Kawase E et al., Int. J. Dev. Biol. 38:p385-390 (1994)に記載されている。ＥＦ細胞は、日本クレア社より購入したB57BL/6jを用いたfibroblastの初代培養により得た。neo耐性ＥＦ細胞は、Itohara S et al., Cell. 72:p337-348 (1998)におけるTCRdeltaノックアウトマウスのヘテロ接合体を用いたfibroblastの初代培養により得た。
(2) ES cell culture The medium described below is used for the culture of ES cells (MS12 strain) and EF cells.
The origin of ES cells (MS12 strain) is described in Kawase E et al., Int. J. Dev. Biol. 38: p385-390 (1994). EF cells were obtained by primary fibroblast culture using B57BL / 6j purchased from CLEA Japan. Neo resistant EF cells were obtained by primary culture of fibroblasts using heterozygotes of TCRdelta knockout mice in Itohara S et al., Cell. 72: p337-348 (1998).
D-MEM (Gibco No.11995-065) 425ml
56℃で45分間不活性化したFCS 75ml
200mM L-glutamino (Gibco No.25030-081) 5ml
Penicillin/Streptomycin (Gibco No.15140-122) 2.5ml
Non-essential amino acids (Gibco No.11140-050) 5ml
Nucleosides 5ml
2-ME (7μl/10 ml PBS) 5ml
LIF (107 u/ml) (ESGRO:Gibco No.13275-029) 50μl
D-MEM (Gibco No.11995-065) 425ml
75 ml FCS inactivated for 45 minutes at 56 ° C
200mM L-glutamino (Gibco No.25030-081) 5ml
Penicillin / Streptomycin (Gibco No.15140-122) 2.5ml
Non-essential amino acids (Gibco No.11140-050) 5ml
Nucleosides 5ml
2-ME (7 μl / 10 ml PBS) 5 ml
LIF (10 7 u / ml) (ESGRO: Gibco No.13275-029) 50μl
ＥＳ細胞及びＥＦ細胞は、0.1％ Gelatinによりコート(室温、１時間以上)したplateにて培養を行う。まずplateにＥＦ細胞をフィーダー細胞として増殖させ、後に液体窒素にて凍結保存してあるＥＳ細胞を37℃の水浴にて溶かし、ＥＦ細胞の上に培養する。ＥＦ細胞及びＥＳ細胞はそれぞれ６×106/150cm2、６×106/50cm2の濃度で培養を開始し、24時間ごとに培地を交換する。 ES cells and EF cells are cultured on plates coated with 0.1% Gelatin (room temperature, 1 hour or longer). First, EF cells are grown as feeder cells on the plate, and then ES cells that have been cryopreserved in liquid nitrogen are dissolved in a 37 ° C. water bath and cultured on the EF cells. EF cells and ES cells each culture initiation at a concentration of 6 × 10 6 / 150cm 2, 6 × 10 6 / 50cm 2, replacing the medium every 24 hours.
（３）エレクトロポレーション
ＥＳ細胞の細胞数が4000万程度になった時点でエレクトロポレーションを行う。
0.05％Trypsin-0.53mM EDTA(Gibco No.25300-054)で37℃にて５分処理をした後、等量の培地を加えサスペンドし、細胞数をカウントする。4000万個分の細胞を遠心し、上清を除去し、ＤＮＡ溶液(下記)を加え、ＰＢＳ(−)にて800μlに液量を調整する。キュベットに溶液を移し、Capacitance＝3μF、Volts＝0.5kVにてエレクトロポレーションを行う。エレクトロポレーション後の細胞液に必要量の培地を加え、500万cells/10cm plateにて８枚のplateに分けて培養を再び行う。Plateには予めneo耐性ＥＦ細胞を前述の方法でフィーダー細胞として増殖させておく。
(3) Electroporation When the number of ES cells reaches about 40 million, electroporation is performed.
After treating with 0.05% Trypsin-0.53 mM EDTA (Gibco No. 25300-054) at 37 ° C. for 5 minutes, add an equal volume of medium, suspend, and count the number of cells. Centrifuge 40 million cells, remove the supernatant, add the DNA solution (below), and adjust the volume to 800 μl with PBS (−). Transfer the solution to a cuvette and perform electroporation at Capacitance = 3 μF and Volts = 0.5 kV. A necessary amount of medium is added to the cell solution after electroporation, and the cells are divided into 8 plates in a 5 million cells / 10 cm plate and cultured again. In the plate, neo-resistant EF cells are previously grown as feeder cells by the method described above.
（４）エレクトロポレーション用のＤＮＡ溶液の調整
ベクタープラスミド 30μg/tube
High buffer 10μl
NotI ３μl(30 U:２〜３倍過剰量)
合計100μl
(4) Preparation of DNA solution for electroporation Vector plasmid 30 μg / tube
High buffer 10μl
NotI 3μl (30 U: 2-3 times excess)
100 μl total
上記混合物を37℃で一晩インキュベートする。エタノール300μｌを加えてよく混合し、15000rpmで5分間遠心する。上清を捨て、ペレットを70％エタノールでリンスする。チューブを培養フード内に静置してエタノールを蒸発させる。600μｌのD-PBSを添加し、DNAペレットを懸濁する。65℃で5分間インキュベートし、よく混合してDNAを溶解させる。 Incubate the mixture at 37 ° C. overnight. Add 300 μl of ethanol, mix well, and centrifuge at 15000 rpm for 5 minutes. Discard the supernatant and rinse the pellet with 70% ethanol. Leave the tube in the culture hood to evaporate the ethanol. Add 600 μl D-PBS and suspend the DNA pellet. Incubate at 65 ° C for 5 minutes and mix well to dissolve the DNA.
（５）G418による選別
エレクトロポレーションの24時間後、培養液にG418を0.15mg/ml加えた選択培地での
培養を開始する。選択培地での培養は一週間ほど行い、G418に対して耐性を持った細胞が形成するコロニーの直径が１mmを越えたものから拾い上げ、Gelatinコート(上記)
した96穴plateにTrypsin処理(上記)後に等量ずつ移し、50％コンフルエント以上になった時点で細胞の凍結(下記)及びＤＮＡの抽出(下記)を行う。
(5) Selection with G418 After 24 hours of electroporation, culture in a selective medium in which 0.15 mg / ml of G418 is added to the culture solution is started. Incubate in selective medium for about a week, pick up colonies with a diameter of more than 1 mm from cells that are resistant to G418, Gelatin coat (above)
After the Trypsin treatment (above), the same amount is transferred to the 96-well plate, and when it becomes 50% confluent or higher, the cells are frozen (below) and DNA is extracted (below).
（６）細胞の凍結
96穴plateに培養したＥＳ細胞に対してTrypsin処理(上記)を行う。血清：DMSO=4:1の割合の溶液を作り、それをTrypsin済み細胞液と等量加え、サスペンドする。ミネラルオイルを静注し、アルミホイルで96穴plateを包み、発砲スチロールのコンテナに入れ、-80℃にて保存する。
(6) Freezing of cells
Trypsin treatment (above) is performed on ES cells cultured in 96-well plates. Make a solution of serum: DMSO = 4: 1, add the same amount of trypsined cell solution, and suspend. Inject mineral oil intravenously, wrap the 96-well plate with aluminum foil, place in a foamed polystyrene container, and store at -80 ° C.
（７）ＤＮＡの抽出
Lysis buffer (0.1M Tris, 5mM EDTA, 0.2％ SDS, 0.2mg/ml Proteinase K、200mM
NaCl)を96穴plateに50μlずつ加え、55℃で一晩置く。次の日、96穴plateのＤＮＡ溶液を1.5mlチューブに移す。さらに100μlの100％エタノールを加え、ボルテックス後、15000rpmで10分遠心(常温)する。上清を廃棄し、80％エタノールで２回洗浄する。ＴＥ(pH=0.8)を30μl加え、スピンダウンし、室温で一晩置く。
(7) DNA extraction
Lysis buffer (0.1M Tris, 5mM EDTA, 0.2% SDS, 0.2mg / ml Proteinase K, 200mM
NaCl) is added to each 96-well plate in a volume of 50 μl and placed at 55 ° C. overnight. The next day, transfer the 96-well plate DNA solution to a 1.5 ml tube. Add 100 μl of 100% ethanol, vortex, and centrifuge at 15000 rpm for 10 minutes (room temperature). Discard the supernatant and wash twice with 80% ethanol. Add 30 μl of TE (pH = 0.8), spin down and leave overnight at room temperature.
（８）ポジティブセレクション
抽出後のＤＮＡはBamHI及びEcoRVで切断し、図１に記載のプローブ(1)、プローブ(2)でSouthern Blottingにて確認する。バンドの位置はBamH Iでの切断の場合、陽性：９kb、陰性：14kbの位置に出る。EcoR Vでの切断の場合、陽性：７kb、陰性11kbの位置に出る。
(8) Positive selection The extracted DNA is cleaved with BamHI and EcoRV, and confirmed by Southern Blotting with the probe (1) and probe (2) shown in FIG. The band positions appear at positions of positive: 9 kb and negative: 14 kb in the case of cleavage with BamHI. In the case of cleavage with EcoR V, the position is positive: 7 kb, negative 11 kb.
（９）インジェクション
相同組換えを起こしていることが確認されたＥＳ細胞を解凍後に培養し、Trypsin処理により分散した。マウスBalb/C系統の雄を掛け合わせた同系統の雌より８細胞期胚を取り出した。この胚から透明帯を外した後、分散した上記ＥＳ細胞を培養させた(20ES細胞／８細胞期胚１個)。これを偽妊娠処理した雌マウスの子宮に移し、胎児の発生を継続させることによりキメラマウスを得た。このキメラマウスの雄を黒色であるC57BL/6jの雌と交配し、生まれてきた仔マウスのうち野ネズミ色(agouti)ではなく黒色(non-agouti)のものを選び、その層の一部を切断した試料から染色体ＤＮＡを抽出した。このＤＮＡ溶液を鋳型としてneoのprimer set (Forward : cttgggtggagaggctattc（配列番号１）, Reverse:aggtgagatgacaggagatc（配列番号２）, Product size=280bp)にて遺伝子変異が全身に導入されたマウスを確認した。
(9) Injection ES cells that were confirmed to have undergone homologous recombination were cultured after thawing and dispersed by trypsin treatment. Eight cell stage embryos were removed from females of the same strain crossed with males of the mouse Balb / C strain. After removing the zona pellucida from the embryo, the dispersed ES cells were cultured (one embryo at 20 ES cell / 8 cell stage). This was transferred to the uterus of a female mouse treated with pseudopregnancy, and the development of the fetus was continued to obtain a chimeric mouse. The male of this chimeric mouse was crossed with a female of C57BL / 6j which is black, and among the born pups, we chose black (non-agouti) instead of wild mouse (agouti), and part of that layer Chromosomal DNA was extracted from the cut sample. Using this DNA solution as a template, mice with a gene mutation introduced into the whole body were confirmed using neo primer set (Forward: cttgggtggagaggctattc (SEQ ID NO: 1), Reverse: agggtgagatgacaggagatc (SEQ ID NO: 2), Product size = 280 bp).
上述の方法により得られたヘテロ接合体同士を交配して、変異caps2遺伝子のホモ接合体を得た。欠失させたCAPS2遺伝子の1st exon内のprimer set (Forward:AAACACTCGGGCGCCGAACT（配列番号３）, Reverse:GACTCCTCCTCGCTGGAAGA（配列番号４）, Product size=200bp)と、先述のneoのprimer setでPCRを行うことで、野生型、ヘテロ接合体、ホモ接合体の決定を行った。 The heterozygotes obtained by the above-described method were crossed to obtain a homozygote of the mutant caps2 gene. Perform PCR using the primer set (Forward: AAACACTCGGGCGCCGAACT (SEQ ID NO: 3), Reverse: GACTCCTCCTCGCTGGAAGA (SEQ ID NO: 4), Product size = 200bp) within the first exon of the deleted CAPS2 gene and the above-mentioned neo primer set. The wild type, heterozygote, and homozygote were determined.
PCR反応液の組成は下記の通りである。
水 13.7μl
10x ExTaq buffer (TakaRa) 2.0μl
dNTP Mix (2.5mM each) 1.6μl
10μM Primer (Forward) 1μl
10μM Primer (Reverse) 1μl
Ex Taq (50U/μl) 0.2μl
ＤＮＡ溶液 0.5μl
(計) 20μl
The composition of the PCR reaction solution is as follows.
Water 13.7μl
10x ExTaq buffer (TakaRa) 2.0μl
dNTP Mix (2.5mM each) 1.6μl
10μM Primer (Forward) 1μl
10μM Primer (Reverse) 1μl
Ex Taq (50U / μl) 0.2μl
DNA solution 0.5 μl
(Total) 20μl
PCR条件は下記の通りである。
初めに、94℃で１分
続いて、94℃で20秒、60℃で20秒、及び72℃で20秒を35サイクル
最後に、72℃で５分
４℃に保持
PCR conditions are as follows.
First, 94 ° C for 1 minute, followed by 35 cycles of 94 ° C for 20 seconds, 60 ° C for 20 seconds, and 72 ° C for 20 seconds Finally, hold at 72 ° C for 5 minutes at 4 ° C
実施例２：ホモ接合体における小脳の形状変化
生後８日目の野生型およびホモ接合体マウスを４％ＰＦＡ(パラホルムアルデヒド)で灌流固定後、脳を取り出し、再度４％ＰＦＡで４時間後固定した。15％ショ糖溶液に４℃で１昼夜浸して置換後、ＯＣＴコンパウンド(サクラ精器(株))に入れて凍結させる。凍結ブロックを14μm厚の切片にクリオスタットで薄切りにする。この切片を野生型とホモ接合体マウスで比較したところ、ホモ接合体マウスで小脳のＶＩ葉とＶＩＩ葉の間の溝が欠失していることが観察された。
Example 2: Change in shape of cerebellum in homozygote Wild type and homozygous mice on the 8th day after birth were fixed by perfusion fixation with 4% PFA (paraformaldehyde), and then the brain was taken out and fixed again after 4 hours with 4% PFA. did. After substituting in a 15% sucrose solution at 4 ° C. for one day, place in an OCT compound (Sakura Seiki Co., Ltd.) and freeze. The frozen block is sliced into 14 μm thick sections with a cryostat. When this section was compared between a wild-type mouse and a homozygous mouse, it was observed that the groove between the cerebellar VI and VII leaves was deleted in the homozygous mouse.
また生後17日目のマウスで同様の処理をしたところ、外顆粒層における細胞数がホモ接合体マウスにおいて多いことが分かった(図２)。これは顆粒細胞の移動の遅滞を示している。 In addition, when the same treatment was performed on mice on the 17th day after birth, the number of cells in the outer granule layer was found to be large in homozygous mice (FIG. 2). This indicates a delay in granule cell migration.
生後８日目の切片で抗カルビンジン抗体(小脳プルキンエ細胞のマーカータンパク質)により免疫染色を行った。上記切片を５％正常ロバ血清で１時間ブロッキングした後、1/1000に希釈した１次抗体(抗カルビンジン抗体)と４℃で一昼夜反応させた。次いで1/1000に希釈した蛍光二次抗体を室温で１時間反応させ、蛍光顕微鏡にて観察した。その結果、プルキンエ細胞の樹状突起の低形成が確認された(図３)。 Immunostaining was performed with an anti-calbindin antibody (marker protein of cerebellar Purkinje cells) on a section on the 8th day after birth. The above sections were blocked with 5% normal donkey serum for 1 hour, and then reacted with a primary antibody (anti-calbindin antibody) diluted 1/1000 at 4 ° C. overnight. Subsequently, the fluorescent secondary antibody diluted to 1/1000 was reacted at room temperature for 1 hour and observed with a fluorescence microscope. As a result, low formation of dendrites of Purkinje cells was confirmed (FIG. 3).
上記の３項目はBDNFノックアウトマウスで知られている事実である。我々は以前の論文でCAPS2タンパク質が小脳においてBDNFやNT-3などの神経栄養因子の分泌に関わっていることを示唆したが、今回のcaps2遺伝子のノックアウトマウスに関しても、in vivoのレベルにおいてもBDNFの分泌を制御していることが示された。 The above three items are facts known for BDNF knockout mice. In our previous paper, we suggested that the CAPS2 protein is involved in the secretion of neurotrophic factors such as BDNF and NT-3 in the cerebellum. However, both the caps2 gene knockout mouse and the in vivo level of BDNF It was shown to regulate the secretion of.
Claims (9)
（１）小脳のＶＩ葉とＶＩＩ葉の間の溝が欠失している。
（２）小脳における顆粒細胞の移動が遅滞しており、外顆粒層における細胞数が多い。
（３）小脳プルキンエ細胞の樹状突起の低形成が見られる。 The knockout non-human mammal or part thereof according to claim 1 or 2, which exhibits at least one of the following findings.
(1) The groove between the cerebellar VI and VII leaves is missing.
(2) The migration of granule cells in the cerebellum is delayed and the number of cells in the outer granule layer is large.
(3) Low formation of dendrites of cerebellar Purkinje cells is observed.
A screening method for a therapeutic / prophylactic agent for a disease associated with an abnormality of a nerve cell, using the knockout non-human mammal according to any one of claims 1 to 7 or a part thereof.
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2004
- 2004-09-17 JP JP2004271208A patent/JP2006081496A/en active Pending
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