JP2014223067A - Psd-zip70 gene knockout non-human animal, and use for the same - Google Patents
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Abstract
Description
本発明は、脳や神経系についての研究の他、癌の研究などに用いる実験動物として有用な、ＰＳＤ−Ｚｉｐ７０遺伝子ノックアウト非ヒト動物、および実験動物などとしてのその用途に関する。 The present invention relates to a PSD-Zip70 gene knockout non-human animal, which is useful as an experimental animal used for research on cancer, in addition to research on the brain and nervous system, and its use as an experimental animal.
ＰＳＤ−Ｚｉｐ７０タンパク質は、本発明者らの研究グループによってラットの脳の神経細胞の樹状突起上のシナプス後肥厚部（ＰＳＤ：Ｐｏｓｔ−Ｓｙｎａｐｔｉｃ Ｄｅｎｓｉｔｙ）分画から単離同定された、例えば配列表の配列番号１のアミノ酸配列（対応するｃＤＮＡ配列は配列表の配列番号２を参照のこと）で表される分子量が約７０ｋＤａであってロイシンジッパードメインとコイルドコイルドメインを持ったタンパク質であり、シナプス形成や神経伝達機能制御に重要な役割を果たしていることが知られている（例えば非特許文献１や非特許文献２を参照のこと）。近年、行動や学習という形で表出する脳や神経系についての研究が盛んに行われていることは一般にもよく知られているところであるが、そうした状況の中、ＰＳＤ−Ｚｉｐ７０遺伝子の機能にも注目が集まっている。また、ＰＳＤ−Ｚｉｐ７０遺伝子は、ヒト染色体８ｐ２２由来の癌抑制遺伝子であるＦｅｚ１／Ｌｚｔｓ１遺伝子と相同であることが知られており、当該遺伝子の機能が明らかになることによって癌の発症の仕組みなどもこれまで以上に明らかになると期待されている。 PSD-Zip70 protein has been isolated and identified from the Post-Synaptic Density (PSD) fraction on dendrites of rat brain neurons by our research group, for example, the sequence listing Is a protein having a molecular weight of about 70 kDa and a leucine zipper domain and a coiled coil domain represented by the amino acid sequence of SEQ ID NO: 1 (for the corresponding cDNA sequence, see SEQ ID NO: 2). It is known to play an important role in controlling neurotransmitter function (for example, see Non-Patent Document 1 and Non-Patent Document 2). In recent years, it is well known that research on the brain and nervous system expressed in the form of behavior and learning is well known, but in this situation, the function of the PSD-Zip70 gene Also attracting attention. The PSD-Zip70 gene is known to be homologous to the Fez1 / Lzts1 gene, which is a tumor suppressor gene derived from human chromosome 8p22, and the mechanism of cancer onset is also revealed by clarifying the function of the gene. It is expected to become clearer than ever.
個体レベルや組織・細胞レベルでＰＳＤ−Ｚｉｐ７０遺伝子の機能を解析する際、当該遺伝子の機能を欠損させた非ヒト動物、例えばＰＳＤ−Ｚｉｐ７０遺伝子ノックアウトマウスを用いた解析が不可欠である。ＰＳＤ−Ｚｉｐ７０遺伝子ノックアウトマウスは、非特許文献３において、高頻度の腫瘍形成の表現型を示すＦｅｚ１／Ｌｚｔｓ１遺伝子ノックアウトマウスとして既に紹介されている。非特許文献３に記載のＰＳＤ−Ｚｉｐ７０（Ｆｅｚ１／Ｌｚｔｓ１）遺伝子ノックアウトマウスは、当該遺伝子が有する全部で３つのエクソン（第１エクソンから第３エクソン）のうちの第１エクソンのみをターゲットとし、第１エクソンの開始コドンの近傍（１２３ｂｐ下流）に終始コドンを導入することで、遺伝子機能の欠損がもたらされるように設計されている。しかしながら、ＰＳＤ−Ｚｉｐ７０タンパク質は、Ｎ末端に脂質修飾を受けて細胞膜へ集積する機能ドメインを持つことから、第１エクソンの開始コドンの近傍に終始コドンを導入する方法では、当該ドメインの部分タンパク質が発現することで、遺伝子機能が完全に欠損されない可能性や発現した部分タンパク質が機能解析に何らかの影響を及ぼす可能性を否定できない。従って、非特許文献３に記載のＰＳＤ−Ｚｉｐ７０遺伝子ノックアウトマウスは、この点において改良の余地を残している。 When analyzing the function of the PSD-Zip70 gene at the individual level or tissue / cell level, analysis using a non-human animal deficient in the function of the gene, such as a PSD-Zip70 gene knockout mouse, is indispensable. The PSD-Zip70 gene knockout mouse has already been introduced in Non-Patent Document 3 as a Fez1 / Lzts1 gene knockout mouse exhibiting a phenotype of high frequency tumor formation. The PSD-Zip70 (Fez1 / Lzts1) gene knockout mouse described in Non-Patent Document 3 targets only the first exon of all three exons (first exon to third exon) of the gene, The gene function is designed to be lost by introducing a stop codon near the start codon of 1 exon (123 bp downstream). However, since PSD-Zip70 protein has a functional domain that is lipid-modified at the N-terminus and accumulates in the cell membrane, in the method of introducing a termination codon near the initiation codon of the first exon, the partial protein of the domain is It cannot be denied that there is a possibility that the gene function is not completely lost or that the expressed partial protein has some influence on the functional analysis. Therefore, the PSD-Zip70 gene knockout mouse described in Non-Patent Document 3 leaves room for improvement in this respect.
そこで本発明は、ＰＳＤ−Ｚｉｐ７０遺伝子の機能を完全に欠損させた、ＰＳＤ−Ｚｉｐ７０遺伝子ノックアウト非ヒト動物、および実験動物などとしてのその用途を提供することを目的とする。 Therefore, an object of the present invention is to provide a use of the PSD-Zip70 gene knockout non-human animal, a laboratory animal, or the like that has completely lost the function of the PSD-Zip70 gene.
上記の点に鑑みてなされた本発明のＰＳＤ−Ｚｉｐ７０遺伝子ノックアウト非ヒト動物は、請求項１記載の通り、ＰＳＤ−Ｚｉｐ７０遺伝子の機能を完全に欠損させた、ＰＳＤ−Ｚｉｐ７０タンパク質を部分的にも全く発現しないことを特徴とする。
また、請求項２記載のＰＳＤ−Ｚｉｐ７０遺伝子ノックアウト非ヒト動物は、請求項１記載のＰＳＤ−Ｚｉｐ７０遺伝子ノックアウト非ヒト動物において、ＰＳＤ−Ｚｉｐ７０遺伝子の第１エクソンから第３エクソンの全てを欠失させたことを特徴とする。
また、本発明は、請求項３記載の通り、請求項１または２記載のＰＳＤ−Ｚｉｐ７０遺伝子ノックアウト非ヒト動物の実験動物としての使用である。
また、請求項４記載の使用は、脳および／または神経系の異常を示すモデル動物としての請求項３記載の使用である。
また、請求項５記載の使用は、請求項４記載の使用において、脳および／または神経系の異常がシナプス形成および／または神経伝達機能制御の異常であることを特徴とする。
また、本発明は、請求項６記載の通り、請求項１または２記載のＰＳＤ−Ｚｉｐ７０遺伝子ノックアウト非ヒト動物由来の脳神経細胞の脳および／または神経系の異常を示すモデル細胞としての使用である。
また、本発明は、請求項７記載の通り、請求項１または２記載のＰＳＤ−Ｚｉｐ７０遺伝子ノックアウト非ヒト動物および／またはこの動物由来の脳神経細胞に対する作用を評価することによる脳および／または神経系の異常の予防・治療薬のスクリーニング方法である。
The PSD-Zip70 gene knockout non-human animal of the present invention made in view of the above-mentioned points partially includes the PSD-Zip70 protein which is completely deficient in the function of the PSD-Zip70 gene as described in claim 1. It is characterized by no expression at all.
Further, the PSD-Zip70 gene knockout non-human animal according to claim 2 is the PSD-Zip70 gene knockout non-human animal according to claim 1, wherein all of the first exon to the third exon of the PSD-Zip70 gene are deleted. It is characterized by that.
Moreover, this invention is use as a laboratory animal of the PSD-Zip70 gene knockout nonhuman animal of Claim 1 or 2 as described in Claim 3.
The use according to claim 4 is the use according to claim 3 as a model animal showing abnormalities in the brain and / or nervous system.
The use according to claim 5 is characterized in that, in the use according to claim 4, the abnormality in the brain and / or nervous system is an abnormality in synapse formation and / or neurotransmitter function control.
Moreover, this invention is use as a model cell which shows the abnormality of the brain and / or nervous system of the cranial nerve cell derived from the PSD-Zip70 gene knockout non-human animal of Claim 1 or 2 as described in Claim 6. .
Further, the present invention provides the brain and / or nervous system by evaluating the action on the non-human animal of the PSD-Zip70 gene knockout according to claim 1 or 2 and / or brain neurons derived from this animal as described in claim 7. This is a screening method for preventive / therapeutic drugs for abnormalities.
本発明によれば、ＰＳＤ−Ｚｉｐ７０遺伝子の機能を完全に欠損させた、ＰＳＤ−Ｚｉｐ７０遺伝子ノックアウト非ヒト動物、および実験動物などとしてのその用途を提供することができる。本発明のＰＳＤ−Ｚｉｐ７０遺伝子ノックアウト非ヒト動物は、ＰＳＤ−Ｚｉｐ７０遺伝子の機能が完全に欠損されており、ＰＳＤ−Ｚｉｐ７０タンパク質を部分的にも全く発現しない。従って、ＰＳＤ−Ｚｉｐ７０タンパク質の部分タンパク質が発現することで、遺伝子機能が完全に欠損されない可能性や発現した部分タンパク質が機能解析に何らかの影響を及ぼす可能性が全くないので、脳や神経系の他、癌などにおけるＰＳＤ−Ｚｉｐ７０遺伝子の機能解明のための優れた実験動物である。 ADVANTAGE OF THE INVENTION According to this invention, the use as a PSD-Zip70 gene knockout non-human animal, the experimental animal, etc. which completely deleted the function of PSD-Zip70 gene can be provided. The PSD-Zip70 gene knockout non-human animal of the present invention is completely deficient in the function of the PSD-Zip70 gene and does not express the PSD-Zip70 protein even partially. Therefore, since the partial protein of PSD-Zip70 protein is expressed, there is no possibility that the gene function is not completely lost or the expressed partial protein has any influence on the functional analysis. It is an excellent experimental animal for elucidating the function of PSD-Zip70 gene in cancer and the like.
本発明のＰＳＤ−Ｚｉｐ７０遺伝子ノックアウト非ヒト動物は、ＰＳＤ−Ｚｉｐ７０遺伝子の機能を完全に欠損させた非ヒト動物、即ち、ＰＳＤ−Ｚｉｐ７０タンパク質をコードする非ヒト動物の内在性遺伝子であるＰＳＤ−Ｚｉｐ７０遺伝子（そのｃＤＮＡ配列としては例えば配列表の配列番号２で表されるものが挙げられるがこの配列に限定されるものではない）に対して破壊や欠失や置換などが行われることで、ＰＳＤ−Ｚｉｐ７０タンパク質を部分的にも全く発現しない非ヒト動物である。なお、本発明における非ヒト動物としては、例えばマウスやラットなどの齧歯目の動物が挙げられる。 The PSD-Zip70 gene knockout non-human animal of the present invention is a PSD-Zip70 that is an endogenous gene of a non-human animal that is completely deficient in the function of the PSD-Zip70 gene, that is, a non-human animal that encodes the PSD-Zip70 protein. By performing destruction, deletion, substitution, etc. on a gene (its cDNA sequence includes, for example, that represented by SEQ ID NO: 2 in the sequence listing, but is not limited to this sequence), PSD -A non-human animal that does not express any part of the Zip70 protein. In addition, examples of the non-human animal in the present invention include rodent animals such as mice and rats.
本発明のＰＳＤ−Ｚｉｐ７０遺伝子ノックアウト非ヒト動物の作製方法は、ＰＳＤ−Ｚｉｐ７０遺伝子の機能を完全に欠損させた非ヒト動物、即ち、ＰＳＤ−Ｚｉｐ７０タンパク質を部分的にも全く発現しない非ヒト動物を作製することができる方法であればどのようなものであってもよいが、例えば、ＰＳＤ−Ｚｉｐ７０遺伝子が有する全部で３つのエクソン（第１エクソンから第３エクソン）を、当該領域をＮｅｏ遺伝子カセットなどのマーカー遺伝子を含むカセットで置換することで欠失させることを目的としたターゲッティングベクターを作製し、作製されたベクターをエレクトロポレーション法によってＥＳ細胞に導入し、相同組み換えを起こしたＥＳ細胞を選択し、選択されたＥＳ細胞系を用いて生殖系列のキメラマウスを作製し、野生型マウスと交配させることによって得られるヘテロ接合体マウス（Ｆ１／雑種第一代）同士を交配させることによって作製することができる。 The method for producing a PSD-Zip70 gene knockout non-human animal of the present invention comprises a non-human animal that is completely deficient in the function of the PSD-Zip70 gene, that is, a non-human animal that does not partially express the PSD-Zip70 protein. Any method can be used as long as it can be prepared. For example, the PSD-Zip70 gene has all three exons (from the first exon to the third exon), and the region is designated as a Neo gene cassette. A targeting vector intended to be deleted by replacement with a cassette containing a marker gene such as the above is prepared, and the prepared vector is introduced into ES cells by electroporation, and the ES cells that have undergone homologous recombination Select and create germline chimeric mice using the selected ES cell line It can be produced by mating between heterozygous mice obtained by crossing with wild-type mice (F1 / F1 hybrid).
本発明のＰＳＤ−Ｚｉｐ７０遺伝子ノックアウト非ヒト動物は、出生から繁殖可能齢までの外見的特徴が野生型非ヒト動物と大差がなく、繁殖能力についても野生型非ヒト動物と同等であるので、例えばＰＳＤ−Ｚｉｐ７０タンパク質が重要な役割を果たしているシナプス形成や神経伝達機能制御に対する個体レベルや組織・細胞レベルでの遺伝子機能の解析を行うための実験動物として有用である。うつ病をはじめとする感情障害や統合失調症などの精神疾患や精神発達遅滞ではシナプス形成の異常がその発症に関わっていることが知られている。従って、本発明のＰＳＤ−Ｚｉｐ７０遺伝子ノックアウト非ヒト動物は、こうした疾患の発症メカニズムの解明や予防・治療法の開発などのための貴重な研究材料となる。また、ＰＳＤ−Ｚｉｐ７０タンパク質は、Ｌａｐｓｅｒ１タンパク質のＳｈａｎｋ３（自閉症関連遺伝子産物）結合ドメインと相同な配列を有しており、自閉症などの発達障害にも関与することが強く示唆されているので、本発明のＰＳＤ−Ｚｉｐ７０遺伝子ノックアウト非ヒト動物は、こうした疾患の発症メカニズムの解明や予防・治療法の開発などのための貴重な研究材料にもなる。また、ＰＳＤ−Ｚｉｐ７０遺伝子は、癌抑制遺伝子としての機能も有するので、本発明のＰＳＤ−Ｚｉｐ７０遺伝子ノックアウト非ヒト動物は、癌の発症メカニズムの解明や予防・治療法の開発などのための貴重な研究材料にもなる。 Since the PSD-Zip70 gene knockout non-human animal of the present invention is not significantly different from the wild-type non-human animal in appearance characteristics from birth to reproductive age, the reproductive ability is equivalent to that of the wild-type non-human animal. It is useful as an experimental animal for analyzing gene functions at the individual level and tissue / cell level for synapse formation and neurotransmission function control in which PSD-Zip70 protein plays an important role. It is known that abnormalities of synapse formation are involved in the development of emotional disorders such as depression, mental disorders such as schizophrenia, and mental retardation. Therefore, the PSD-Zip70 gene knockout non-human animal of the present invention is a valuable research material for elucidating the onset mechanism of such diseases and developing preventive and therapeutic methods. Moreover, PSD-Zip70 protein has a sequence homologous to the Shank3 (autism-related gene product) binding domain of the Lapper1 protein, and is strongly suggested to be involved in developmental disorders such as autism. Therefore, the PSD-Zip70 gene knockout non-human animal of the present invention is also a valuable research material for elucidating the onset mechanism of such diseases and developing preventive / therapeutic methods. In addition, since the PSD-Zip70 gene also has a function as a tumor suppressor gene, the PSD-Zip70 gene knockout non-human animal of the present invention is valuable for elucidating the onset mechanism of cancer and developing preventive / therapeutic methods. It can be used as research material.
以下、本発明をＰＳＤ−Ｚｉｐ７０遺伝子ノックアウトマウスの実施例によって詳細に説明するが、本発明は以下の記載に限定して解釈されるものではない。 Hereinafter, the present invention will be described in detail by way of examples of PSD-Zip70 gene knockout mice, but the present invention should not be construed as being limited to the following description.
（Ａ）本発明のＰＳＤ−Ｚｉｐ７０遺伝子ノックアウトマウスの作製
１．ＰＳＤ−Ｚｉｐ７０遺伝子近傍のゲノムＤＮＡのクローニング
ＰＳＤ−Ｚｉｐ７０遺伝子の第１エクソンの上流域８．２ｋｂと第３エクソンの下流域１．９５ｋｂの領域のそれぞれのゲノムＤＮＡ断片を、Ｃ５７ＢＬ／６Ｊ系統由来のマウスゲノムＤＮＡのＢＡＣライブラリーの陽性クローンからそれぞれクローニングした。
(A) Production of PSD-Zip70 gene knockout mouse of the present invention Cloning of genomic DNA in the vicinity of PSD-Zip70 gene The genomic DNA fragments of the upstream region of 8.2 kb of the first exon and the downstream region of 1.95 kb of the third exon of the PSD-Zip70 gene were derived from the C57BL / 6J strain. Each was cloned from a positive clone of a BAC library of mouse genomic DNA.
２．ＰＳＤ−Ｚｉｐ７０ターゲッティングベクターの構築（図１）
ＰＳＤ−Ｚｉｐ７０ターゲッティングベクターは、第１エクソンの上流域８．２ｋｂｐと第３エクソンの下流域１．９５ｋｂｐの領域のそれぞれに対する相同配列の間に、ホスホグリセリン酸キナーゼ（ＰＧＫ）プロモーターによって発現されるネオマイシン耐性遺伝子カセット（Ｎｅｏ遺伝子カセット）をＰＳＤ−Ｚｉｐ７０遺伝子がコードされている方向に対して逆向きに挿入することによって作製した。
2. Construction of PSD-Zip70 targeting vector (Figure 1)
The PSD-Zip70 targeting vector is a neomycin expressed by a phosphoglycerate kinase (PGK) promoter between homologous sequences for each of the region of 8.2 kbp upstream of the first exon and 1.95 kbp downstream of the third exon. A resistance gene cassette (Neo gene cassette) was prepared by inserting in a direction opposite to the direction in which the PSD-Zip70 gene was encoded.
３．ＥＳ細胞への相同組み換え
制限酵素を用いてこのＰＳＤ−Ｚｉｐ７０ターゲッティングベクターを直鎖状とし、エレクトロポレーション法によってＣ５７ＢＬ／６ＮＴａｃ系統由来ＥＳ細胞（ｉＴＬ ＩＣ１細胞）に導入した。ネオマイシン耐性ＥＳ細胞クローンをＧ４１８によって選択し、ＰＣＲ法によって相同組み換え体を選別した。ＰＣＲ法による相同組み換えの確認は、図１に示したネオマイシン耐性遺伝子カセット配列に対して作製したプライマーＮ１（配列表の配列番号３）と、３’側相同配列よりも下流領域の配列に対して作製したプライマーＡ２（配列表の配列番号４）を用い、相同組み換えが起こった場合に増幅される２．２ｋｂの断片を指標にして行った。図２にＥＳ細胞クローンの選別結果の一例を示す。図２から明らかなように、相同組み換えが起こったことを示す２．２ｋｂの断片が増幅されたＥＳ細胞クローンの存在が確認できた（クローン６２１，６２５，６２６，６４６）。
3. Homologous recombination into ES cells This PSD-Zip70 targeting vector was linearized using a restriction enzyme and introduced into C57BL / 6NTac line-derived ES cells (iTL IC1 cells) by electroporation. Neomycin resistant ES cell clones were selected by G418 and homologous recombinants were selected by PCR. Confirmation of homologous recombination by the PCR method is performed on the primer N1 (SEQ ID NO: 3 in the sequence listing) prepared for the neomycin resistance gene cassette sequence shown in FIG. Using the prepared primer A2 (SEQ ID NO: 4 in the sequence listing), a 2.2 kb fragment amplified when homologous recombination occurred was used as an index. FIG. 2 shows an example of ES cell clone selection results. As is clear from FIG. 2, the presence of ES cell clones in which a 2.2-kb fragment indicating that homologous recombination occurred was confirmed (clone 621, 625, 626, 646).
４．組み換えＥＳ細胞の個体への導入および組み換えマウスの作製
相同組み換えが起こったＥＳ細胞クローン（クローン６２５）を用い、文献（Ａ．Ｌ．Ｊｏｙｎｅｒ，Ｇｅｎｅ Ｔａｒｇｅｔｉｎｇ：Ａ ｐｒａｃｔｉｃａｌ ａｐｐｒｏａｃｈ，１ｓｔ ｅｄｉｔｉｏｎ，１９９３）の方法に準じて胚盤胞期のＢａｌｂ／ｃ系統由来の受精卵に対してマイクロインジェクションによって導入し、これを擬妊娠したＢａｌｂ／ｃ系統仮親の子宮内に移植した。作出されたキメラマウスのうち、キメラ細胞頻度が高いオス個体を用いてＢａｌｂ／ｃメス個体と交配してその仔の体色判別することより、生殖細胞にＰＳＤ−Ｚｉｐ７０遺伝子相同組み換えの起こった生殖系列オスキメラ個体を選別した。この選別された生殖系列オスキメラマウスをＣ５７ＢＬ／６Ｊのメスマウスと交配することによりヘテロ接合体マウス（Ｆ１／雑種第一代）を作出し、次いでホモ接合体マウスを得るためにこのヘテロ接合体マウス間で交配を行い、メンデルの法則に従って本発明のＰＳＤ−Ｚｉｐ７０遺伝子ノックアウトマウスを作製した。
4). Introduction of Recombinant ES Cells into Individuals and Production of Recombinant Mice Using the ES cell clone (clone 625) in which homologous recombination has occurred, the method of the literature (AL Joyner, Gene Targeting: A practical approach, 1st edition, 1993) The fertilized eggs derived from the Balb / c strain at the blastocyst stage were introduced by microinjection and transferred into the womb of pseudoparental Balb / c strain foster parents. Among the created chimeric mice, male individuals with high chimera cell frequency are mated with Balb / c female individuals and their pups are discriminated so that their reproductive cells undergo reproductive PSD-Zip70 gene homologous recombination. Lineage male chimera individuals were selected. This selected germline male chimeric mouse is crossed with a C57BL / 6J female mouse to produce a heterozygous mouse (F1 / hybrid first generation), and then to obtain a homozygous mouse, this heterozygous mouse The mice were crossed and PSD-Zip70 gene knockout mice of the present invention were prepared according to Mendel's law.
（Ｂ）（Ａ）で作製した本発明のＰＳＤ−Ｚｉｐ７０遺伝子ノックアウトマウスがＰＳＤ−Ｚｉｐ７０遺伝子およびタンパク質を発現していないことの確認
１．ＰＳＤ−Ｚｉｐ７０の遺伝子型の確認
同腹子として出生したマウスの尾よりゲノムＤＮＡを抽出し、ＥＳ細胞選別の際に用いたプライマーＮ１とプライマーＡ２を用いてＰＣＲを行った。上記の通り、ＰＳＤ−Ｚｉｐ７０遺伝子を欠失した染色体を持つ場合には２．２ｋｂの断片が増幅される。また、ＰＳＤ−Ｚｉｐ７０遺伝子の第３エクソン内の配列に対して作製したプライマーＺ１（配列表の配列番号５）とプライマーＡ２を用いてＰＣＲを行った。この場合、野生型のＰＳＤ−Ｚｉｐ７０遺伝子が残存する染色体を持つ場合には２．１ｋｂの断片が増幅される。図３にこれらのＰＣＲ法による遺伝子型特定の結果を示す。
(B) Confirmation that the PSD-Zip70 gene knockout mouse of the present invention prepared in (A) does not express PSD-Zip70 gene and protein Confirmation of genotype of PSD-Zip70 Genomic DNA was extracted from the tail of a mouse born as a litter and PCR was performed using the primer N1 and primer A2 used for ES cell selection. As described above, a 2.2 kb fragment is amplified when the chromosome lacks the PSD-Zip70 gene. PCR was performed using primer Z1 (SEQ ID NO: 5 in the sequence listing) and primer A2 prepared for the sequence within the third exon of the PSD-Zip70 gene. In this case, when the wild type PSD-Zip70 gene has a remaining chromosome, a 2.1 kb fragment is amplified. FIG. 3 shows the results of genotyping by these PCR methods.
２．ＰＳＤ−Ｚｉｐ７０ｍＲＮＡの発現の確認
同腹子として出生した１２週齢の野生型マウス（ＰＳＤ−Ｚｉｐ７０＋／＋）および本発明のＰＳＤ−Ｚｉｐ７０遺伝子ノックアウトマウス（ＰＳＤ−Ｚｉｐ７０−／−）から得られた大脳皮質組織を用いてＲＴ−ＰＣＲ法によりＰＳＤ−Ｚｉｐ７０ｍＲＮＡの発現を確認した。大脳皮質組織よりｔｏｔａｌＲＮＡを抽出し、それを鋳型としてランダムプライマーを用いた逆転写を行ってｃＤＮＡを合成した。このｃＤＮＡを鋳型としてＰＳＤ−Ｚｉｐ７０遺伝子のコード領域に対して作製したプライマーを用いたＲＴ−ＰＣＲを行い、ＰＳＤ−Ｚｉｐ７０ｍＲＮＡの発現を確認した。陽性対照はＧＡＰＤＨ遺伝子およびβ−アクチン遺伝子を用いた。結果を図４に示す。図４から明らかなように、本発明のＰＳＤ−Ｚｉｐ７０遺伝子ノックアウトマウスでは、ＰＳＤ−Ｚｉｐ７０ｍＲＮＡの発現を確認することができなかった。
2. Confirmation of the expression of PSD-Zip70 mRNA Cerebral cortex obtained from 12-week-old wild-type mice (PSD-Zip70 + / +) born as litters and PSD-Zip70 gene knockout mice (PSD-Zip70 − / −) of the present invention Using tissue, the expression of PSD-Zip70 mRNA was confirmed by RT-PCR. Total RNA was extracted from cerebral cortex tissue, and cDNA was synthesized by reverse transcription using random primers as a template. Using this cDNA as a template, RT-PCR was performed using primers prepared for the coding region of the PSD-Zip70 gene, and the expression of PSD-Zip70 mRNA was confirmed. As a positive control, GAPDH gene and β-actin gene were used. The results are shown in FIG. As is clear from FIG. 4, the expression of PSD-Zip70 mRNA could not be confirmed in the PSD-Zip70 gene knockout mouse of the present invention.
３．ＰＳＤ−Ｚｉｐ７０タンパク質の発現の確認
同腹子として出生した８週齢の野生型マウス（ＰＳＤ−Ｚｉｐ７０＋／＋）および本発明のＰＳＤ−Ｚｉｐ７０遺伝子ノックアウトマウス（ＰＳＤ−Ｚｉｐ７０−／−）から得られた大脳皮質組織を用いてウエスタンブロット解析を行った。大脳皮質組織は１００倍容量のＳＤＳサンプルバッファーで溶解し、各５μＬを用いてアクリルアミドゲル電気泳動によって展開し、ＰＶＤＦ膜上に転写を行った。１次抗体としては抗ＰＳＤ−Ｚｉｐ７０特異抗体（ウサギ産生ポリクローナル抗体）、２次抗体としてＨＲＰ標識抗ウサギ抗体（ヤギ産生ポリクロ―ナル抗体）を用い、化学発光法によって検出した。陽性対照はβ−アクチンタンパク質およびＴｕｊ１（神経細胞特異的チューブリンβ３）タンパク質をそれぞれの特異抗体を用いて検出した。結果を図５に示す。図５から明らかなように、本発明のＰＳＤ−Ｚｉｐ７０遺伝子ノックアウトマウスでは、ＰＳＤ−Ｚｉｐ７０タンパク質の発現を確認することができなかった。
3. Confirmation of expression of PSD-Zip70 protein Cerebrum obtained from 8-week-old wild-type mice (PSD-Zip70 + / +) and PSD-Zip70 gene knockout mice (PSD-Zip70 − / −) of the present invention born as litters Western blot analysis was performed using cortical tissue. Cerebral cortical tissue was dissolved in a 100-fold volume of SDS sample buffer, developed by acrylamide gel electrophoresis using 5 μL of each, and transferred onto a PVDF membrane. Anti-PSD-Zip70 specific antibody (rabbit-produced polyclonal antibody) was used as the primary antibody, and HRP-labeled anti-rabbit antibody (goat-produced polyclonal antibody) was used as the secondary antibody, which was detected by the chemiluminescence method. As positive controls, β-actin protein and Tuj1 (nerve cell-specific tubulin β3) protein were detected using respective specific antibodies. The results are shown in FIG. As is clear from FIG. 5, expression of PSD-Zip70 protein could not be confirmed in the PSD-Zip70 gene knockout mouse of the present invention.
４．まとめ
以上の結果から、本来、ＰＳＤ−Ｚｉｐ７０タンパク質が豊富に発現する大脳皮質において、本発明のＰＳＤ−Ｚｉｐ７０遺伝子ノックアウトマウスは、ＰＳＤ−Ｚｉｐ７０の発現がｍＲＮＡとタンパク質いずれにおいても消失していることが確認できた。
4). Summary From the above results, it can be seen that PSD-Zip70 gene knockout mice of the present invention, in the cerebral cortex that abundantly express PSD-Zip70 protein, have lost expression of PSD-Zip70 in both mRNA and protein. It could be confirmed.
（Ｃ）（Ａ）で作製した本発明のＰＳＤ−Ｚｉｐ７０遺伝子ノックアウトマウスの外見的特徴と繁殖能力
出生から繁殖可能齢までの外見的特徴は野生型マウスと大差がなく、繁殖能力についても野生型マウスと同等であった。
(C) Appearance characteristics and reproductive ability of the PSD-Zip70 gene knockout mouse of the present invention prepared in (A) The appearance characteristics from birth to reproductive age are not much different from those of wild-type mice. It was equivalent to a mouse.
（Ｄ）（Ａ）で作製した本発明のＰＳＤ−Ｚｉｐ７０遺伝子ノックアウトマウス由来の脳神経細胞を用いたＰＳＤ−Ｚｉｐ７０遺伝子の欠失によるシナプス形成や神経伝達機能制御への影響の検討（脳神経細胞の電気生理学的変化）
８週齢の野生型マウス（ＰＳＤ−Ｚｉｐ７０＋／＋）および本発明のＰＳＤ−Ｚｉｐ７０遺伝子ノックアウトマウス（ＰＳＤ−Ｚｉｐ７０−／−）から得られた大脳皮質組織を用いて電気生理学的解析を行った。具体的には、Ｙａｓｕｄａ Ｈ．ｅｔ ａｌ．，Ｎａｔｕｒｅ Ｎｅｕｒｏｓｃｉｅｎｃｅ ２００３；６（１）：１５−１６に記載の方法に準じ、大脳皮質組織から厚さ４００μｍの急性脳スライス標本を作製し、前部帯状回皮質の第２／３層の錐体細胞に対して１μＭテトドロトキシンと１００μＭピクロトキシンの存在下でホールセルパッチクランプ法によって膜電位−７５ｍＶで固定して微小興奮性シナプス後電流を計測した。微小興奮性シナプス後電位は、振幅がシナプス後部の機能的な神経伝達物質の存在量、頻度は機能的なシナプスの形成密度あるいはシナプス前部のシナプス小胞の開口頻度を反映し、これらの減少はシナプス形成や神経伝達機能制御の異常によって起こる。結果を図６に示す。図６から明らかなように、本発明のＰＳＤ−Ｚｉｐ７０遺伝子ノックアウトマウスは、微小興奮性シナプス後電流の振幅については野生型マウスと大差がなかったが、頻度については野生型マウスに比較して有意な減少が認められた（野生型マウス：ｎ＝１２，ＰＳＤ−Ｚｉｐ７０遺伝子ノックアウトマウス：ｎ＝１５，ｐ＜０．０５）。以上の結果から、本発明のＰＳＤ−Ｚｉｐ７０遺伝子ノックアウトマウス由来の脳神経細胞は、ＰＳＤ−Ｚｉｐ７０遺伝子の欠失によるシナプス形成や神経伝達機能制御の異常に基づく脳や神経系の異常を示すモデル細胞として、こうした異常の、メカニズムの解明や、電気生理学的変化に対する程度の緩和や改善を指標とした予防・治療薬のスクリーニングなどに利用することができることがわかった。
(D) Examination of the effect on the synapse formation and the control of neurotransmitter function by the deletion of PSD-Zip70 gene using the brain neurons derived from the PSD-Zip70 gene knockout mouse of the present invention prepared in (A) (electricity of brain neurons) Physiological changes)
Electrophysiological analysis was performed using cerebral cortex tissues obtained from 8-week-old wild type mice (PSD-Zip70 + / +) and PSD-Zip70 gene knockout mice of the present invention (PSD-Zip70 − / −). Specifically, Yasuda H. et al. et al. , Nature Neuroscience 2003; 6 (1): 15-16, an acute brain slice specimen having a thickness of 400 μm is prepared from a cerebral cortex tissue, and a 2/3 layer cone of the anterior cingulate cortex The cells were fixed at a membrane potential of -75 mV by whole cell patch clamp method in the presence of 1 μM tedotrotoxin and 100 μM picrotoxin, and the microexcitatory post-synaptic current was measured. Microexcitatory post-synaptic potential decreases in amplitude, with the amount of functional neurotransmitter present in the postsynaptic area, frequency reflecting the density of functional synapse formation or the frequency of presynaptic vesicle opening. Is caused by abnormalities in synapse formation and control of neurotransmitter function. The results are shown in FIG. As is clear from FIG. 6, the PSD-Zip70 gene knockout mouse of the present invention was not significantly different from the wild type mouse in the amplitude of the microexcitatory post-synaptic current, but the frequency was significant compared to the wild type mouse. Reduction was observed (wild-type mice: n = 12, PSD-Zip70 gene knockout mice: n = 15, p <0.05). From the above results, the cranial nerve cells derived from the PSD-Zip70 gene knockout mouse of the present invention are model cells showing abnormalities in the brain and nervous system based on abnormal synapse formation and neurotransmitter function control due to deletion of the PSD-Zip70 gene. It was found that it can be used to elucidate the mechanism of such abnormalities and to screen prophylactic / therapeutic drugs using the degree of mitigation or improvement of electrophysiological changes as an index.
（Ｅ）（Ａ）で作製した本発明のＰＳＤ−Ｚｉｐ７０遺伝子ノックアウトマウスを用いたＰＳＤ−Ｚｉｐ７０遺伝子の欠失による空間作業記憶や不安傾向への影響の検討
１．空間作業記憶への影響
８〜１０週齢の野生型マウス（ＰＳＤ−Ｚｉｐ７０＋／＋）および本発明のＰＳＤ−Ｚｉｐ７０遺伝子ノックアウトマウス（ＰＳＤ−Ｚｉｐ７０−／−）のそれぞれについて、Ｙ字迷路を用いて行動特性を評価した。具体的には、Ｓａｒｔｅｒ Ｍ．ｅｔ ａｌ．，Ｐｓｙｃｈｏｐｈａｒｍａｃｏｌｏｇｙ（Ｂｅｒｌ）．１９８８；９４（４）：４９１−４９５に記載の方法に準じ、長さと幅が等しい３本のアームが等角度に配置された構造を有するＹ字迷路の中心にマウスを置いて５分間自由に移動させた。マウスの移動をビデオカメラによって撮影し、時間内にアームに進入した回数と順番を記録した。マウスがアームに進入した回数から１引いた値を自発運動量、３回連続で異なるアームに進入した回数を自発運動量の値で除した数値を交替量（ａｌｔｅｒｎａｔｉｏｎ）として算出した。交替量は直前に入ったアームの記憶を短期的に保持する空間作業記憶の能力を反映する。結果を図７に示す。図７から明らかなように、本発明のＰＳＤ−Ｚｉｐ７０遺伝子ノックアウトマウスは、時間内の自発運動量については野生型マウスと大差がなかったが、交替量については野生型マウスに比較して有意な減少（空間作業記憶の能力が障害）が認められた（野生型マウス：ｎ＝１８，ＰＳＤ−Ｚｉｐ７０遺伝子ノックアウトマウス：ｎ＝２０，ｐ＜０．０５）。
(E) Examination of the influence on the spatial working memory and anxiety tendency by the deletion of the PSD-Zip70 gene using the PSD-Zip70 gene knockout mouse of the present invention prepared in (A) Effects on spatial working memory For each of 8-10 week old wild type mice (PSD-Zip70 + / +) and PSD-Zip70 gene knockout mice of the present invention (PSD-Zip70 − / −), the Y-shaped maze was used. Behavioral characteristics were evaluated. Specifically, Sarter M.M. et al. , Psychopharmacology (Bell). 1988; 94 (4): 491-495, the mouse is placed in the center of a Y-shaped maze having a structure in which three arms having equal lengths and widths are arranged at equal angles, and is freely allowed for 5 minutes. Moved. The movement of the mouse was photographed with a video camera, and the number and order of entering the arm in time were recorded. A value obtained by dividing the value obtained by subtracting 1 from the number of times the mouse entered the arm was divided by the amount of spontaneous exercise, and the number of times the mouse entered the different arm three times in succession was calculated as the alternation. The amount of replacement reflects the ability of the spatial working memory to hold the memory of the arm that entered immediately before. The results are shown in FIG. As is clear from FIG. 7, the PSD-Zip70 gene knockout mouse of the present invention was not significantly different from the wild-type mouse in the amount of spontaneous movement in time, but the amount of alternation was significantly decreased compared to the wild-type mouse. (Capability of spatial working memory was impaired) (wild type mice: n = 18, PSD-Zip70 gene knockout mice: n = 20, p <0.05).
２．不安傾向への影響（その１）
８〜１０週齢の野生型マウス（ＰＳＤ−Ｚｉｐ７０＋／＋）および本発明のＰＳＤ−Ｚｉｐ７０遺伝子ノックアウトマウス（ＰＳＤ−Ｚｉｐ７０−／−）のそれぞれについて、オープンフィールド試験による行動特性を評価した。具体的には、Ｗａｌｓｈ ＲＮ．ｅｔ ａｌ．，Ｐｓｙｃｈｏｌｏｇｉｃａｌ Ｂｕｌｌｅｔｉｎ １９７６；８３（３）：４８２−５０４に記載の方法に準じ、高さ２０ｃｍの壁で囲われた６０ｃｍ四方の正方形の容器の中心にマウスを置いて５分間自由に移動させた。マウスの移動をビデオカメラによって撮影し、時間内に容器内で移動した軌跡を記録した。容器の中心から３０ｃｍ四方の区域を中央部とした。マウスは壁際に沿って移動する傾向が強いが、本能的な探索行動によって身を隠すことができない中央部にも進入する。中央部への進入回数が少ないほど強い不安を反映していると判断することができる。結果を図８に示す。図８から明らかなように、本発明のＰＳＤ−Ｚｉｐ７０遺伝子ノックアウトマウスは、時間内の総移動距離については野生型マウスと大差がなかったが、中央部への進入回数については野生型マウスに比較して有意な減少（不安傾向の増強）が認められた（野生型マウス：ｎ＝１２，ＰＳＤ−Ｚｉｐ７０遺伝子ノックアウトマウス：ｎ＝１４，ｐ＜０．０５）。
2. Impact on anxiety (part 1)
The behavioral characteristics by the open field test were evaluated for each of 8 to 10 weeks old wild type mice (PSD-Zip70 + / +) and PSD-Zip70 gene knockout mice of the present invention (PSD-Zip70 − / −). Specifically, Walsh RN. et al. In accordance with the method described in S., Psychological Bulletin 1976; 83 (3): 482-504, a mouse was placed in the center of a 60 cm square container surrounded by a 20 cm high wall and moved freely for 5 minutes. The movement of the mouse was photographed with a video camera, and the trajectory moved in the container in time was recorded. An area 30 cm square from the center of the container was taken as the center. The mouse has a strong tendency to move along the wall, but also enters the center where it cannot be hidden by instinctive search behavior. It can be determined that the less the number of times of entering the center, the stronger the anxiety is reflected. The results are shown in FIG. As is clear from FIG. 8, the PSD-Zip70 gene knockout mouse of the present invention was not significantly different from the wild-type mouse in terms of the total movement distance in time, but compared with the wild-type mouse in terms of the number of entry into the center. A significant decrease (enhanced anxiety tendency) was observed (wild-type mice: n = 12, PSD-Zip70 gene knockout mice: n = 14, p <0.05).
３．不安傾向への影響（その２）
８〜１０週齢の野生型マウス（ＰＳＤ−Ｚｉｐ７０＋／＋）および本発明のＰＳＤ−Ｚｉｐ７０遺伝子ノックアウトマウス（ＰＳＤ−Ｚｉｐ７０−／−）のそれぞれについて、高架十字迷路を用いて行動特性を評価した。具体的には、Ｌｉｓｔｅｒ ＲＧ．，Ｐｓｙｃｈｏｐｈａｒｍａｃｏｌｏｇｙ（Ｂｅｒｌ）．１９８７；９２（２）：１８０−１８５に記載の方法に準じ、高さ５０ｃｍの場所に長さと幅が等しい４本のアームが全体で十字を形成するように配置され、向かい合う２本のアームは側面を高さ１５ｃｍの壁に囲まれているが、それに直交する２本のアームは側面に壁がない構造を有する高架十字迷路の中心にマウスを置いて５分間自由に移動させた。マウスの移動をビデオカメラによって撮影し、時間内に高架十字迷路内で移動した軌跡を記録した。側面に壁がないアームは高架となっているため、マウスは落下に対する不安を感じるので、そこでの運動量が少ないほど強い不安を反映していると判断することができる。結果を図９に示す。図９から明らかなように、本発明のＰＳＤ−Ｚｉｐ７０遺伝子ノックアウトマウスは、時間内の側面に壁がないアームでの運動量が野生型マウスと比較して有意に少なかった（不安傾向の増強）（野生型マウス：ｎ＝１３，ＰＳＤ−Ｚｉｐ７０遺伝子ノックアウトマウス：ｎ＝１５，ｐ＜０．０５）。
3. Impact on anxiety (part 2)
The behavioral characteristics of each of 8-10 week old wild type mice (PSD-Zip70 + / +) and PSD-Zip70 gene knockout mice of the present invention (PSD-Zip70 − / −) were evaluated using the elevated plus maze. Specifically, Lister RG. , Psychopharmacology (Bell). 1987; 92 (2): 180-185, four arms having the same length and width are arranged at a height of 50 cm so as to form a cross as a whole. Although the side was surrounded by a wall of 15 cm in height, the two arms perpendicular to it were moved freely for 5 minutes by placing the mouse in the center of the elevated plus maze having a structure with no wall on the side. The movement of the mouse was photographed with a video camera, and the trajectory that moved in the elevated plus maze in time was recorded. Since the arm without a wall on the side is elevated, the mouse feels anxiety about the fall, so it can be determined that the less the momentum there is, the stronger the anxiety is reflected. The results are shown in FIG. As is clear from FIG. 9, the PSD-Zip70 gene knockout mouse of the present invention had significantly less momentum in the arm with no wall on the side in time than the wild type mouse (enhanced anxiety tendency) ( Wild type mouse: n = 13, PSD-Zip70 gene knockout mouse: n = 15, p <0.05).
４．まとめ
以上の結果から、本発明のＰＳＤ−Ｚｉｐ７０遺伝子ノックアウトマウスは、ＰＳＤ−Ｚｉｐ７０遺伝子の欠失により、空間作業記憶の能力が障害されているとともに、不安傾向が増強されていることから、シナプス形成や神経伝達機能制御の異常に基づく脳や神経系の異常を示すモデル動物として、こうした異常の、メカニズムの解明や、空間作業記憶の能力の障害や不安傾向の増強に対する程度の緩和や改善を指標にした予防・治療薬のスクリーニングなどに利用することができることがわかった。
4). Conclusion From the above results, the PSD-Zip70 gene knockout mouse of the present invention has impaired spatial working memory ability due to deletion of the PSD-Zip70 gene and has an increased anxiety tendency. As a model animal that shows abnormalities in the brain and nervous system based on abnormalities in the control of neurotransmitter functions and neurotransmitters, indicators of elucidation of the mechanism of these abnormalities, relaxation and improvement of the degree of spatial working memory ability impairment and anxiety tendency enhancement It was found that it can be used for screening preventive and therapeutic drugs.
本発明は、ＰＳＤ−Ｚｉｐ７０遺伝子の機能を完全に欠損させた、ＰＳＤ−Ｚｉｐ７０遺伝子ノックアウト非ヒト動物、および実験動物などとしてのその用途を提供することができる点において産業上の利用可能性を有する。 INDUSTRIAL APPLICABILITY The present invention has industrial applicability in that it can provide its use as a PSD-Zip70 gene knockout non-human animal, a laboratory animal, or the like that is completely deficient in the function of the PSD-Zip70 gene. .
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