JP2003102332A - MOUSE EXPRESSING MUTANT HUMAN tau GENE - Google Patents
MOUSE EXPRESSING MUTANT HUMAN tau GENEInfo
- Publication number
- JP2003102332A JP2003102332A JP2002176601A JP2002176601A JP2003102332A JP 2003102332 A JP2003102332 A JP 2003102332A JP 2002176601 A JP2002176601 A JP 2002176601A JP 2002176601 A JP2002176601 A JP 2002176601A JP 2003102332 A JP2003102332 A JP 2003102332A
- Authority
- JP
- Japan
- Prior art keywords
- lys
- gly
- ser
- pro
- ala
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、変異型ヒトタウタ
ンパク質を発現するマウスに関し、詳しくは、変異型ヒ
トタウタンパク質を少なくとも脳において発現してい
る、学習能低下を示すマウスに関する。TECHNICAL FIELD The present invention relates to a mouse that expresses a mutant human tau protein, and more particularly to a mouse that expresses a mutant human tau protein in at least the brain and exhibits reduced learning ability.
【０００２】[0002]
【従来の技術】動・植物細胞に広く存在する外径約25ｎ
Ｍの管状構造物である微小管は、有糸***や、細胞内物
質・小器官の輸送等に関与しており、神経細胞中では軸
策輸送を担っている。微小管は、α−チューブリン（分
子量57,000）とβ−チューブリン（分子量53,000）が管
状に重合することにより構成されており、脱重合したサ
ブユニットと常に平衡状態にある。微小管の重合・脱重
合は、GTP及び微小管結合タンパク質（microtubule-ass
ociated proteins：MAPs）と総称される非チューブリン
タンパク質の働きにより制御されている。2. Description of the Related Art Approximately 25n outer diameter widely existing in animal and plant cells
Microtubules, which are the tubular structures of M, are involved in mitosis, transport of intracellular substances and organelles, etc., and are responsible for axonal transport in nerve cells. Microtubules are composed of tubular polymerization of α-tubulin (molecular weight 57,000) and β-tubulin (molecular weight 53,000) and are always in equilibrium with depolymerized subunits. Polymerization / depolymerization of microtubules is performed by GTP and microtubule-binding protein (microtubule-ass
It is regulated by the action of non-tubulin proteins collectively called ociated proteins (MAPs).
【０００３】タウタンパク質（τ protein）はMAPsの一
つであり、サブユニットの重合を促して微小管の形成を
促進すると共に、微小管を束ねる機能を行っている。タ
ウタンパク質は、成人脳では部分的にリン酸化された状
態で存在するが、リン酸化されたタウはリン酸化されて
いないタウに比して微小管重合促進能が低いことが判明
している。[0003] Tau protein (τ protein) is one of MAPs, which promotes the polymerization of subunits to promote the formation of microtubules and also has the function of bundling microtubules. Although tau protein exists in a partially phosphorylated state in the adult brain, it has been found that phosphorylated tau has a lower ability to promote microtubule polymerization than tau that is not phosphorylated.
【０００４】ヒトのタウタンパク質のゲノム構造は、10
0 kbp超のＤＮＡよりなっており、染色体17q21.8上に16
個のコード領域として組織されている。最初のコード領
域は、エキソン−１又はエキソン０と呼ばれるが、翻訳
されない。エキソン１、４、５、７、９、１１、１２及
び１３／１４は、神経組織中では恒常的に発現されてい
るが、エクソン２、３、４Ａ、６、８及び１０は、異な
ったタウタンパク質アイソフォームの産生を、部分的に
担っている。成人皮質では、エクソン４Ａ、６及び８は
発現されず、エクソン２、３及び１０は選択的スプライ
シングを受けて、アミノ酸個数352〜441個の６種の明確
に異なったタウタンパク質アイソフォームを与える。こ
れら６種のアイソフォームは、Ｃ末端領域おいて31又は
32個のアミノ酸が３回（３リピートタウ）又は４回(４
リピートタウ：エクソン１０を発現)の何れの連続的繰
り返しを有するかということと、Ｎ末端領域において29
個（エクソン２に対応）若しくは58個（エクソン２＋３
に対応）のアミノ酸の何れが挿入されているか又はそれ
らの挿入が無いか、ということとによって異なってい
る。The genomic structure of human tau protein is 10
It is composed of more than 0 kbp of DNA and is 16 on chromosome 17q21.8.
It is organized as individual code regions. The first coding region, called exon-1 or exon 0, is not translated. Exons 1, 4, 5, 7, 9, 11, 12 and 13/14 are constitutively expressed in neural tissue, whereas exons 2, 3, 4A, 6, 8 and 10 are different tau. Partly responsible for the production of Protein Isoforms. In the adult cortex, exons 4A, 6 and 8 are not expressed and exons 2, 3 and 10 are alternatively spliced to give 6 distinct tau protein isoforms of amino acids 352-441. These 6 isoforms have 31 or
32 amino acids 3 times (3 repeat tau) or 4 times (4
Repeat tau: which exon 10 is expressed) and which N-terminal region it has.
Number of pieces (corresponding to exon 2) or 58 pieces (exon 2 + 3)
(Corresponding to) and whether or not they have been inserted.
【０００５】胎児から幼若期では、エクソン２、３及び
１０に対応するアミノ酸配列を何れも有しない最も小さ
いアイソフォームのみが発現しているが、その後の成熟
と共に、より大きなアイソフォームが発現するようにな
り、成人では４リピート（4R）タウが著増することが知
られている。From the fetus to the juvenile stage, only the smallest isoform having no amino acid sequences corresponding to exons 2, 3 and 10 is expressed, but with subsequent maturation, a larger isoform is expressed. It is known that 4 repeat (4R) tau is significantly increased in adults.
【０００６】脳の神経細胞死を伴う高度の進行性痴呆を
引き起こす疾患であるアルツハイマー病では、患者脳の
神経細胞中のタウタンパク質のリン酸化が亢進が認めら
れると共に、同病における脳の神経細胞に神経原線維変
化（neurofibrillary tangle）が認められる。神経原線
維変化は、嗜銀性線維構造物の存在として認められる
が、その微細構造は、平行に走る、枝分かれのない多数
の線維からなる。各線維はＰＨＦ（paired helical fil
aments）と呼ばれており、その基本骨格は、過剰にリン
酸化されたタウであることが判明している。このことか
ら、タウの過剰なリン酸化とそれによる微小管重合能の
低下、細胞骨格の崩壊、軸策輸送の障害等により、ニュ
ーロンの変性・脱落が生じることが、アルツハイマー病
発症の一因であると考えられている。[0006] In Alzheimer's disease, which is a disease that causes a high degree of progressive dementia with nerve cell death in the brain, phosphorylation of tau protein in nerve cells of the patient's brain is observed to be enhanced, and at the same time, brain nerve cells in the disease Neurofibrillary tangles are observed in the. Neurofibrillary tangles are seen as the presence of argyrophilic fibrous structures, but their microstructure consists of a large number of parallel, unbranched fibers. Each fiber is PHF (paired helical fil)
aments), whose basic skeleton has been found to be hyperphosphorylated tau. From this fact, degeneration and loss of neurons due to excessive phosphorylation of tau and the resulting decrease in microtubule polymerization ability, disruption of cytoskeleton, impaired axonal transport, etc. are one of the causes of the onset of Alzheimer's disease. Is believed to be.
【０００７】また、タウタンパク質は、アルツハイマー
病以外にもいくつかの神経変性病に関与していることが
知られている。例えば、FTDP-17（frontotemporal deme
ntiawith parkinsonism linked to chromosome 17)（本
明細書では「染色体17関連パーキンソン症候群随伴前頭
側頭部痴呆」と訳す。) と呼ばれる神経変性疾患におい
て、アルツハイマー病の患者に見られるのと類似のタウ
タンパク質を含む線維状の集合物が患者の脳から見つか
っており、更に、FTDP-17の原因がタウの遺伝子変異に
あることが明らかとなっている。また、ピック病（Pic
k's disease）、進行性核上性麻痺（progressive supra
nuclear palsy）、大脳皮質基底核神経節変性症（corti
cobasal degeneration）についても、タウタンパク質の
異常が関与するものと考えられている。Further, tau protein is known to be involved in several neurodegenerative diseases other than Alzheimer's disease. For example, FTDP-17 (frontotemporal deme
ntiawith parkinsonism linked to chromosome 17) (translated herein as "chromosome 17-related Parkinsonism-associated frontotemporal dementia"), a tau protein similar to that found in patients with Alzheimer's disease Fibrous aggregates containing these have been found in the brains of patients, and it has been clarified that the cause of FTDP-17 is a tau gene mutation. In addition, Pick disease (Pic
k's disease), progressive supra
nuclear palsy), corticobasal ganglion degeneration (corti
cobasal degeneration) is also considered to be involved in tau protein abnormalities.
【０００８】[0008]
【発明が解決しようとする課題】本発明は、タウタンパ
ク質異常を伴うニューロン疾患に対する治療剤を開発す
るために用いる道具としてのモデル動物を作成すること
を目的とする。そのようなニューロン疾患としては、ア
ツルハイマー病、FTDP-17、ピック病（Pick's diseas
e）、進行性核上性麻痺（progressive supranuclear pa
lsy）、大脳皮質基底核神経節変性症（corticobasal de
generation）が挙げられる。The object of the present invention is to prepare a model animal as a tool used for developing a therapeutic agent for a neuronal disease associated with tau protein abnormality. Such neurological diseases include Azulheimer's disease, FTDP-17, Pick's diseas
e), progressive supranuclear pa
lsy), basal ganglia ganglion degeneration (corticobasal de
generation).
【０００９】[0009]
【課題を解決するための手段】本発明者らは、エクソン
２及び３を欠きクソン１０を有するアイソフォーム（本
明細書において「０Ｎ４Ｒアイソフォーム」ともい
う。）の野生型ヒトタウ遺伝子、又はFTDP-17を有する
ヒト患者に見られる変異を導入することにより作成した
同アイソフォームの変異型ヒトタウ遺伝子をマウスに組
み込んで、得られたトランスジェニックマウスの学習能
力を観察し評価すると共にその基礎にある歯状回の電気
生理学的変化を検討した。組み込んだ該変異型ヒトタウ
遺伝子によりコードされるアミノ酸配列においては、最
長アイソフォーム（本明細書において「２Ｎ４Ｒアイソ
フォーム」ともいう。その野生型アミノ酸配列を、配列
表において配列番号６で示す。）の野生型ヒトタウタン
パク質の第279番目のアミノ酸に相当する位置のアミノ
酸が、アスパラギンからリシンに置換されている。この
アミノ酸置換は、野生型ヒトタウタンパク質の２Ｎ４Ｒ
アイソフォームをコードするＤＮＡ（配列表において配
列番号５に示す。）の第837番目のヌクレオチドに相当
する位置のヌクレオチドをチミン（T）からグアニン
（G）へと置換することにより行った。得られた野生
型、変異型ヒトタウ遺伝子組み込みマウスのそれぞれに
おいて、対応するヒトタウ遺伝子が脳の各部位において
発現していること、対照マウス（ヒトタウ遺伝子を組み
込まず）及び野生型ヒトタウ遺伝子組み込みマウスに比
して、変異型ヒトタウ遺伝子組み込みマウスにおいて学
習能の低下が健著であること、また、ヒトタウ遺伝子
（変異型、野生型）組み込みマウスにおけるヒトタウの
リン酸化が非組み込みマウスにおけるマウスタウのリン
酸化に比して亢進していること、及び、変異型ヒトタウ
遺伝子組み込みマウスにおける導入ヒトタウのリン酸化
が野生型ヒトタウ遺伝子組み込みマウスにおける導入ヒ
トタウに比して亢進していることを見出した。本発明
は、これらの知見に基づき、なされたものである。Means for Solving the Problems The present inventors have found that a wild-type human tau gene of an isoform lacking exons 2 and 3 and having exon 10 (also referred to herein as “0N4R isoform”), or FTDP-. Incorporating a mutant human tau gene of the same isoform created by introducing a mutation found in a human patient having 17 into the mouse, observing and evaluating the learning ability of the resulting transgenic mouse and evaluating the underlying tooth The electrophysiological changes of the gyrus were examined. In the amino acid sequence encoded by the mutant human tau gene incorporated, the longest isoform (also referred to as “2N4R isoform” in the present specification. Its wild-type amino acid sequence is shown in SEQ ID NO: 6 in the sequence listing). The amino acid at the position corresponding to the 279th amino acid of wild-type human tau protein was replaced with lysine from asparagine. This amino acid substitution corresponds to the 2N4R of wild-type human tau protein.
It was carried out by substituting thymine (T) for guanine (G) at the nucleotide at the position corresponding to the 837th nucleotide in the DNA encoding the isoform (shown in SEQ ID NO: 5 in the sequence listing). In each of the obtained wild-type and mutant-type human tau gene-incorporated mice, the corresponding human tau gene is expressed in each part of the brain, compared to control mice (without the human tau gene-incorporated) and wild-type human tau gene-incorporated mice. In addition, the decrease in learning ability in the mutant human tau gene-incorporated mouse was significant, and the phosphorylation of human tau in the mouse in which the human tau gene (mutant type or wild-type) was incorporated was higher than that in the non-integrated mouse. It was found that the phosphorylation of the introduced human tau in the mutant human tau gene-incorporated mouse was enhanced as compared with the introduced human tau in the wild-type human tau gene-incorporated mouse. The present invention has been made based on these findings.
【００１０】すなわち本発明は、エクソン２及び３の何
れに対応するアミノ酸配列をも有しないがエクソン１０
に対応するアミノ酸を有するアイソフォーム（０Ｎ４Ｒ
アイソフォーム）のヒトタウタンパク質であって、該ヒ
トタウタンパク質において野生型ヒトタウタンパク質の
最長アイソフォーム（２Ｎ４Ｒアイソフォーム）におけ
る第279番目のアミノ酸に相当する位置のアミノ酸がリ
シンであることを特徴とする変異型ヒトタウタンパク質
をコードする遺伝子を組み込んでなり、該変異型ヒトタ
ウタンパク質を少なくとも脳において発現しているトラ
ンスジェニックマウスを提供する。That is, although the present invention has no amino acid sequence corresponding to either exon 2 or 3,
Having an amino acid corresponding to (0N4R
Isoform) human tau protein, wherein the amino acid at the position corresponding to the 279th amino acid in the longest isoform of wild-type human tau protein (2N4R isoform) is lysine. The present invention provides a transgenic mouse in which a gene encoding the mutant human tau protein is incorporated, which expresses the mutant human tau protein in at least the brain.
【００１１】上記変異型ヒトタウは、配列表において配
列番号４に示すアミノ酸配列を有する。従って、本発明
は、該変異型ヒトタウタンパク質が、配列表において配
列番号４に示すアミノ酸配列を有するものである、請求
項１のトランスジェニックマウスをも提供する。The above mutant human tau has the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing. Therefore, the present invention also provides the transgenic mouse according to claim 1, wherein the mutant human tau protein has the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing.
【００１２】該トランスジェニックマウスに導入すべき
変異型ヒトタウ遺伝子は、配列表において配列番号４で
示すアミノ酸配列を有するタンパク質をコードするもの
であればよく、そのような変異型ヒトタウ遺伝子のヌク
レオチド配列は、遺伝子コードの縮重に関する知識を利
用して適宜に決定できる。従って本発明は、該遺伝子
が、配列表において配列番号４に示すアミノ酸配列を有
するタンパク質をコードするヌクレオチド配列を有する
ものである、請求項２のトランスジェニックマウスをも
提供する。The mutant human tau gene to be introduced into the transgenic mouse may be any one as long as it encodes a protein having the amino acid sequence represented by SEQ ID NO: 4 in the sequence listing, and the nucleotide sequence of such mutant human tau gene is , Can be appropriately determined by utilizing the knowledge about the degeneracy of the genetic code. Therefore, the present invention also provides the transgenic mouse according to claim 2, wherein the gene has a nucleotide sequence encoding a protein having the amino acid sequence represented by SEQ ID NO: 4 in the sequence listing.
【００１３】特に、該変異型ヒトタウ遺伝子は、該変異
型ヒトタウタンパク質をコードするＤＮＡにおいて、ヌ
クレオチド663（２Ｎ４ＲアイソフォームＤＮＡのヌク
レオチド837に対応する。）をグアニンとすることによ
り得ることができる。従って、本発明は、該変異型ヒト
タウタンパク質をコードするＤＮＡにおいて、野生型ヒ
トタウタンパク質の最長アイソフォームをコードするＤ
ＮＡの第837番目のヌクレオチドに相当する位置のヌク
レオチドがグアニンであることを特徴とする、請求項１
ないし３の何れかのトランスジェニックマウスをも提供
する。In particular, the mutant human tau gene can be obtained by using nucleotide 663 (corresponding to nucleotide 837 of 2N4R isoform DNA) as guanine in the DNA encoding the mutant human tau protein. Accordingly, the present invention provides a D encoding the longest isoform of wild-type human tau protein in the DNA encoding the mutant human tau protein.
The nucleotide at the position corresponding to the 837th nucleotide of NA is guanine.
Also provided is the transgenic mouse of any one of 1 to 3.
【００１４】特に、該変異型ヒトタウタンパク質は、配
列表において配列番号３に示すヌクレオチド配列を有す
るものであることが好ましい。従って本発明は、該変異
型ヒトタウタンパク質をコードするＤＮＡが、配列表に
おいて配列番号３に示すヌクレオチド配列を有するもの
である、請求項１のトランスジェニックマウスをも提供
する。In particular, the mutant human tau protein preferably has the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing. Therefore, the present invention also provides the transgenic mouse according to claim 1, wherein the DNA encoding the mutant human tau protein has the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing.
【００１５】また本発明のトランスジェニックマウス
は、該変異型ヒトタウタンパク質をコードする遺伝子を
ヘテロで有するもの及びホモで有するものを包含する。The transgenic mouse of the present invention includes those having a gene encoding the mutant human tau protein in a heterozygote and those having a homozygous gene.
【００１６】本発明により、正常マウスに比して学習能
の低下したトランスジェニックマウスを得ることができ
る。該トランスジェニックマウスでは、ヒトのアルツハ
イマー病におけると同様のタウタンパク質リン酸化亢進
及び学習能低下を示す。このことから、本発明のトラン
スジェニックマウスは、アルツハイマー病、FTDP‐17等
による痴呆その他の治療薬の開発のための薬物試験シス
テムとして利用することができる。According to the present invention, it is possible to obtain a transgenic mouse whose learning ability is lower than that of a normal mouse. The transgenic mouse exhibits the same enhanced tau protein phosphorylation and decreased learning ability as in human Alzheimer's disease. From this, the transgenic mouse of the present invention can be used as a drug test system for the development of therapeutic agents for Alzheimer's disease, dementia caused by FTDP-17 and the like.
【００１７】[0017]
【発明の実施の形態】本発明においてマウスに導入して
発現させる変異型ヒトタウタンパク質は、配列表の配列
番号４に示すアミノ酸配列を有する。該アミノ酸配列
は、対応する０Ｎ４Ｒアイソフォームの野生型ヒトタウ
タンパク質の有するアミノ酸配列（配列番号２に示
す。）において、アミノ酸221のアスパラギン（Asn）が
リシン（Lys）で置換されたものである。該アミノ酸221
は、最長のアイソフォームである２Ｎ４Ｒアイソフォー
ムのアミノ酸279に相当する。BEST MODE FOR CARRYING OUT THE INVENTION The mutant human tau protein introduced into a mouse and expressed in the present invention has an amino acid sequence shown in SEQ ID NO: 4 in the sequence listing. The amino acid sequence is the amino acid sequence of the corresponding 0N4R isoform of wild-type human tau protein (shown in SEQ ID NO: 2) in which asparagine (Asn) at amino acid 221 is replaced with lysine (Lys). The amino acid 221
Corresponds to amino acid 279 of the longest isoform, the 2N4R isoform.
【００１８】このアミノ酸置換は、マウスに導入する０
Ｎ４Ｒアイソフォームのヒトタウタンパク質をコードす
るＤＮＡにおいて、野生型（配列番号１）におけるヌク
レオチド663のチミン（T）をグアニン（G）に置換する
ことによって行うことができる（配列番号３）。該ヌク
レオチド663は、２Ｎ４Ｒアイソフォームをコードする
ＤＮＡにおけるヌクレオチド837に相当する。このヌク
レオチド置換は、０Ｎ４Ｒアイソフォーム野生型ヒトタ
ウタンパク質をコードするＤＮＡにおいて、ヌクレオチ
ド663に対して、慣用の方法を用いて部位特異的突然変
異を誘導することによって、容易に得ることができる。This amino acid substitution is introduced into the mouse.
This can be done by substituting guanine (G) for thymine (T) at nucleotide 663 in the wild type (SEQ ID NO: 1) in the DNA encoding the human tau protein of N4R isoform (SEQ ID NO: 3). The nucleotide 663 corresponds to nucleotide 837 in the DNA encoding the 2N4R isoform. This nucleotide substitution can be readily obtained by inducing a site-directed mutation at nucleotide 663 in the DNA encoding the 0N4R isoform wild-type human tau protein using conventional methods.
【００１９】以下実施例を参照して本発明を更に詳細に
説明するが、本発明が該実施例に限定されることは意図
しない。The present invention will be described in more detail with reference to the following examples, but it is not intended that the present invention be limited to the examples.
【００２０】[0020]
【実施例】１．組み込み用野生型ヒトタウタンパク質遺
伝子の作製
ヒトタウ遺伝子として、アミノ酸383個よりなる０Ｎ４
Ｒアイソフォームの野生型ヒトタウタンパク質をコード
するｃＤＮＡ（配列番号１に示す）を含んだＤＮＡ断片
を、大阪市立大学医学部 老年医学研究部門 脳・神経
系分野 森 啓教授より入手した。[Example] 1. Preparation of wild-type human tau protein gene for integration 0N4 consisting of 383 amino acids as human tau gene
A DNA fragment containing a cDNA encoding the wild-type human tau protein of R isoform (shown in SEQ ID NO: 1) was obtained from Professor Kei Mori of the Department of Geriatrics, Department of Geriatrics, Osaka City University School of Medicine.
【００２１】このヒトタウｃＤＮＡを含むＤＮＡ断片を
pET-23dベクター（Novagen）のNco1-EcoR1部位に挿入、
tau-wild/pET-23d（図１）を作製した。Tau-wild/pET-2
3dをNco1消化、断端をクレノウ（Klenow）ＤＮＡポリメ
ラーゼにて平滑末端化した後、Xho1消化してヒトタウｃ
ＤＮＡ（383 アミノ酸をコード）を含むＤＮＡ断片を切
り出し、pGEX-4T-1ベクター（Amersham-Pharmacia）のS
ma1-Xho1部位に挿入し、tau-wild/pGEX-4T（図２）を作
製した。A DNA fragment containing this human tau cDNA is
Inserted into the Nco1-EcoR1 site of pET-23d vector (Novagen),
tau-wild / pET-23d (Fig. 1) was prepared. Tau-wild / pET-2
3d was digested with Nco1, the ends were made blunt with Klenow DNA polymerase, and then digested with Xho1 to give human tau c.
A DNA fragment containing DNA (encoding 383 amino acids) was excised, and S of pGEX-4T-1 vector (Amersham-Pharmacia) was excised.
It was inserted into the ma1-Xho1 site to prepare tau-wild / pGEX-4T (Fig. 2).
【００２２】２．組み込み用変異型タウ遺伝子（T837G
変異）の作製
ヒト変異体タウ断片（病原性変異 N279K）は、Yasuda M
et al., A mutationin the microtubule-associated p
rotein tau in pallido-nigro-luysian degeneration,
Neurology, 1999 Sep 11;53(4):864-8に報告されてい
る。該ＤＮＡ配列は、最長アイソフォーム２Ｎ４Ｒを基
準として数えた837番目の塩基TがGに変異したもの（T83
7G変異）である。同変異を有する０Ｎ４Ｒアイソフォー
ムをコードする遺伝子を次のようにして作製した。すな
わち、Quick change Site-Directed mutagenesis Kit
(Stratagene)を用いて、上記tau-wild/pGEX-4Tに、ｃＤ
ＮＡの第663番目のヌクレオチドであるチミン（T）のグ
アニン（G）による置換を導入した。得られたtau-T837G
/pGEX-4Tを図３に示す。その際、変異導入のために用い
たプライマーは、tauT837G-5:5'-GGCGGGAAGGTGCAGATAAT
TAAGAAGAAGCTGGATCTTAGC（配列番号７）とtauT837G-3:
5'-GCTAAGATCCAGCTTCTTCTTAATTATCTGCACCTTCCCGCC（配
列番号８）である。2. Mutant tau gene for integration (T837G
Mutation) The human mutant tau fragment (pathogenic mutation N279K) was produced by Yasuda M
et al., A mutation in the microtubule-associated p
rotein tau in pallido-nigro-luysian degeneration,
Neurology, 1999 Sep 11; 53 (4): 864-8. The DNA sequence was obtained by mutating the base T at the 837th position counted from the longest isoform 2N4R to G (T83
7G mutation). A gene encoding the 0N4R isoform having the same mutation was prepared as follows. That is, Quick change Site-Directed mutagenesis Kit
(Stratagene), using the above tau-wild / pGEX-4T, cD
A substitution of thymine (T), the 663rd nucleotide of NA, with guanine (G) was introduced. The obtained tau-T837G
/ pGEX-4T is shown in FIG. At that time, the primer used for introducing the mutation was tauT837G-5: 5'-GGCGGGAAGGTGCAGATAAT.
TAAGAAGAAGCTGGATCTTAGC (SEQ ID NO: 7) and tauT837G-3:
5'-GCTAAGATCCAGCTTCTTCTTAATTATCTGCACCTTCCCGCC (SEQ ID NO: 8).
【００２３】３．組み込み用ＤＮＡの修飾
次いで、tau-wild/pGEX-4TのヒトタウｃＤＮＡの開始コ
ドンの5’側にXhoIサイトを入れるために以下の操作を
行った。すなわち、tau-wild/pGEX-4Tを鋳型として、2
つのプライマーtauXho-ATG-5:5'-GTGGATCCCTCGAGCCCATG
GCTGAGCCCCGCCAGGAGTT（配列番号９）とtau419-3:5'-GA
GTCGACTTGTCATCGCTTCCAGTCCC（配列番号１０）にてPCR
を行い、得られたPCR産物をBamHIとPmaCIで消化し、tau
-wild/pGEX-4TのBamHI-PmaCI部位に挿入し、tau-XhoI-w
ild/pGEX-4T（図4）を作製した。また、tau-XhoI-wild/
pGEX-4TのNcoI-SalI部位をtau-T837G/pGEX-4TのNcoI-Sa
lI部位と入れ替えることによって、tau-XhoI-T837G/pGE
X-4T（図5）も作製した。3. Modification of DNA for integration Next, the following operation was performed to insert an XhoI site 5 ′ to the start codon of human tau cDNA of tau-wild / pGEX-4T. That is, using tau-wild / pGEX-4T as a template, 2
Two primers tauXho-ATG-5: 5'-GTGGATCCCTCGAGCCCATG
GCTGAGCCCCGCCAGGAGTT (SEQ ID NO: 9) and tau419-3: 5'-GA
PCR with GTCGACTTGTCATCGCTTCCAGTCCC (SEQ ID NO: 10)
The resulting PCR product was digested with BamHI and PmaCI and tau
-wild / pGEX-4T inserted into BamHI-PmaCI site, tau-XhoI-w
ild / pGEX-4T (Fig. 4) was prepared. Also, tau-XhoI-wild /
The NcoI-SalI site of pGEX-4T was replaced with the NcoI-Sa of tau-T837G / pGEX-4T.
By exchanging with lI site, tau-XhoI-T837G / pGE
X-4T (Fig. 5) was also prepared.
【００２４】４．発現ベクター
組み込みに用いる発現ベクターMoPrP.Xho（Johns Hopki
ns大学より分与）は、Westaway D et al., Neuron, 7:5
9-68(1991), Fischer M et al., The EMBO Journal, 15
(6):1255-1264(1996), 及びBorchelt DR et al., Genet
Anal. 1996 Dec;13(6):159-163に従って、次のように
してcos6.I/LnJ-4からphgPrPを経由して構築することが
できる。
（１）cos6.I/LnJ-4の作製 （Westaway D et al., Neur
on, 7:59-68(1991)を参照）：Westaway D et al., Neur
on, 7:59-68(1991)の記載に従い、Scott M., et al., C
ell, 59:847-857(1989)の方法により、pcos6EMBLベクタ
ー（ATCC No. 37571）と宿主大腸菌NM554株を用いてク
ローンI/LnJ-4の単離が行われる。NM554株のrecA-遺伝
子型は紫外線照射（Stratalinker, Stratagen, San Die
go, CA, USA）に対する抵抗性が対照recA+大腸菌株より
増大していることにより確認される。クローンI/LnJ-4
は、マウスゲノムライブラリーよりPrn-Pオープンリー
ディングフレーム（配列番号１１）及びマウスプリオン
エキソン２（配列番号１２）をプローブに用いて同時ス
クリーニングを行うことにより単離される。コロニーか
ら精製したI/LnJ-4クローンをインビトロパッケージン
グし、大腸菌DH1株に導入し収率を高めることができ
る。4. Expression vector used for integration of expression vector MoPrP.Xho (Johns Hopki
ns University), Westaway D et al., Neuron, 7: 5
9-68 (1991), Fischer M et al., The EMBO Journal, 15
(6): 1255-1264 (1996), and Borchelt DR et al., Genet.
According to Anal. 1996 Dec; 13 (6): 159-163, it can be constructed from cos6.I / LnJ-4 via phgPrP as follows. (1) Preparation of cos6.I / LnJ-4 (Westaway D et al., Neur
on, 7: 59-68 (1991)): Westaway D et al., Neur
on, 7: 59-68 (1991), Scott M., et al., C.
ell, 59: 847-857 (1989), clone I / LnJ-4 is isolated using the pcos6EMBL vector (ATCC No. 37571) and the host Escherichia coli NM554 strain. NM554 strain recA of - genotypes UV irradiation (Stratalinker, Stratagen, San Die
resistance to the control recA + E. coli strain is confirmed. Clone I / LnJ-4
Is isolated from a mouse genomic library by performing a simultaneous screening using the Prn-P open reading frame (SEQ ID NO: 11) and mouse prion exon 2 (SEQ ID NO: 12) as probes. I / LnJ-4 clones purified from colonies can be packaged in vitro and introduced into E. coli strain DH1 to increase the yield.
【００２５】（２）の作製：phgPrPは、 Fischer M et
al., The EMBO Journal, 15(6):1255-1264(1996)に従
い、cos6.I/LnJ-4から作製することができる（Fischer
M et al.（上記）より転載の図６を参照。）。すなわ
ち、まずプリオンタンパク質エキソン１及び２を含んで
なるベクターであるcos6.I/LnJ-4からの7.6ｋｂのXbaI
断片をpBluescriptのXbaI部位に挿入して、pPrPS4を作
製する。NMRIマウスの全脳からのｃＤＮＡをTfiI−EcoR
Iで消化して得られる1.4ｋｂの断片を更にKpnIで消化し
て1.3ｋｂの断片を作製する。この断片と、pPrPS4から
の３ｋｂKpnI−BamHI断片とを、脱リン酸化した、BamHI
及びEcoRIで切断したpBluescript（Stratagene）に連結
することにより、pPrPEli1E24R1を作製する。cos6.I/Ln
J-4からの5.5ｋｂNotI−BspEI断片と、pPrPEli1E24R1か
らの3.5ｋｂNarI−BspEI断片と、そしてpNZW BamHIの３
ｋｂNarI−SalI断片とを、NotI及びSalIで切断し脱リン
酸化したpBluescriptにライゲートすることにより、phg
PrPを作製する。Preparation of (2): phgPrP was prepared by Fischer M et
It can be prepared from cos6.I / LnJ-4 according to al., The EMBO Journal, 15 (6): 1255-1264 (1996) (Fischer
See Figure 6, reproduced from M et al. (Supra). ). That is, first, a 7.6 kb XbaI from cos6.I / LnJ-4, which is a vector containing prion proteins exons 1 and 2
The fragment is inserted into the XbaI site of pBluescript to create pPrPS4. CDNA from whole brain of NMRI mouse was transferred to TfiI-EcoR
The 1.4 kb fragment obtained by digesting with I is further digested with KpnI to prepare a 1.3 kb fragment. This fragment and the 3 kb KpnI-BamHI fragment from pPrPS4 were dephosphorylated, BamHI
And pBluescript (Stratagene) cut with EcoRI to prepare pPrPEli1E24R1. cos6.I / Ln
A 5.5 kb NotI-BspEI fragment from J-4, a 3.5 kb NarI-BspEI fragment from pPrPEli1E24R1, and 3 of pNZW BamHI.
The kbNarI-SalI fragment was ligated to pBluescript cleaved with NotI and SalI and dephosphorylated to give phg
Create PrP.
【００２６】（３）MoPrP.Xhoの作製：MoPrP.Xho は、p
hgPrPから、Borchelt DR et al., Genet Anal. 1996 De
c;13(6):159-163の記載に従い、次のように行われる。B
orchelt DR et al.(上記)より転載の図７を参照。
（Ａ）は、pBluescript KS+ プラスミドにおけるphgPrP
ゲノミックフラグメントのマップであり、ボックスはエ
キソン配列であり、白ボックスは非コード5’配列、黒
ボックスはコード配列、ハッチを入れたボックスは非コ
ード3’配列を表す。図７（Ｂ）に示すように、phgPrP
に加える最初の修飾は、3’SalI部位を除去してNotI部
位で置き換えることにより、マイクロインジェクション
の前のプラスミド配列の切り出しを容易にすることであ
る。phgPrPプラスミドをSalIで消化し、クレノウ（Klen
ow）断片で塞ぎ、NotIリンカーとライゲートし、次いで
NotIで消化し、XhoI部位を壊しておいたpBluescript中
にクローニングする。続いて、図７（Ｃ）に示すよう
に、pBluescript（アンチセンス）の配列に対して及び
オープンリーディングフレーム終止コドンの直ぐ下流の
配列（センス、GGGGTACCTCGAGCCTTCCTGCTTGTTCC：配列
番号１３）に対してそれぞれ相補的なオリゴヌクレオチ
ドプライマー（矢印）を用いて、3’末端の非翻訳配列
をＰＣＲにより増幅する。このセンスプライマーはま
た、連続したKpnI及びXhoI制限酵素部位を、PrP関連配
列より前にコードしている。ＰＣＲ産物をKpnI及びNarI
で切断し、次いで、KpnIとNarIで切断しておいたphgPr
P.NotIにライゲートする。完全に組み立てられた発現プ
ラスミドMoPrP.Xhoを図７（Ｄ）に示す。(3) Preparation of MoPrP.Xho: MoPrP.Xho is p
From hgPrP, Borchelt DR et al., Genet Anal. 1996 De
c; 13 (6): 159-163, as described below. B
See Figure 7 reproduced from orchelt DR et al. (above).
(A) PhgPrP in pBluescript KS + plasmid.
It is a map of genomic fragments, boxes are exon sequences, white boxes represent non-coding 5'sequences, black boxes represent coding sequences, and hatched boxes represent non-coding 3'sequences. As shown in FIG. 7 (B), phgPrP
The first modification to be made is to remove the 3'SalI site and replace it with a NotI site to facilitate excision of the plasmid sequence prior to microinjection. The phgPrP plasmid was digested with SalI and the Klenow (Klen
ow) fragment, ligated with NotI linker, then
Digest with NotI and clone into pBluescript with the XhoI site destroyed. Then, as shown in FIG. 7 (C), it is complementary to the pBluescript (antisense) sequence and to the sequence immediately downstream of the open reading frame stop codon (sense, GGGGTACCTCGAGCCTTCCTGCTTGTTCC: SEQ ID NO: 13), respectively. The 3'end untranslated sequences are amplified by PCR using oligonucleotide primers (arrows). This sense primer also encodes a continuous KpnI and XhoI restriction enzyme site before the PrP-related sequence. PCR product is KpnI and NarI
And then KpnI and NarI.
Ligate to P.NotI. The fully assembled expression plasmid MoPrP.Xho is shown in Figure 7 (D).
【００２７】５．トランスジェニックマウスの作成
tau-XhoI-wild/pGEX-4T及びtau-XhoI-T837G/pGEX-4T
を、それぞれXhoI消化することにより、ヒトタウｃＤＮ
Ａ(アミノ酸383残基)を含むＤＮＡ断片を切り出し、MoP
rPベクターのXhoI部位に挿入後、NotI消化を行い、導入
遺伝子（マウスプリオンプロモーター付きＤＮＡ断片ta
u-wild/MoPrP及びtau-T837G/MoPrP）を、それぞれ切り
出した。切り出したtau-wild/MoPrP、tau-T837G/MoPrP
は、5 ng/ml の濃度になるように マイクロインジェク
ション緩衝液 (1mM Tris-HCl, 0.1mM EDTA, pH 8.0)に
溶解した。5. Creation of transgenic mice tau-XhoI-wild / pGEX-4T and tau-XhoI-T837G / pGEX-4T
Were digested with XhoI to obtain human tau-cDN
A DNA fragment containing A (383 amino acid residues) was cut out and subjected to MoP
After insertion into the XhoI site of the rP vector, digestion with NotI was performed, and the transgene (mouse prion promoter-attached DNA fragment ta
u-wild / MoPrP and tau-T837G / MoPrP) were each cut out. Cut out tau-wild / MoPrP, tau-T837G / MoPrP
Was dissolved in a microinjection buffer (1 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) to a concentration of 5 ng / ml.
【００２８】C57BL/6とSJL F2との交配により、交雑群
を作成した。これら交雑群同士の交配より得た受精卵を
改良ホイッテン（Whitten）培養液（ＮａＣｌ 514ｍｇ
／ｄｌ、ＫＣｌ 36.0ｍｇ／ｄｌ、ＫＨ2ＰＯ4 16.0ｍ
ｇ／ｄｌ、ＭｇＳＯ4・７Ｈ2Ｏ 29.0ｍｇ／ｄｌ、Ｎａ
ＨＣＯ3 190.0ｍｇ／ｄｌ、グルコース 100.0ｍｇ／ｄ
ｌ、ピルビン酸ナトリウム 3.5ｍｇ／ｄｌ、乳酸カルシ
ウム・５Ｈ2Ｏ 53.0ｍｇ／ｄｌ、ストレプトマイシン
5.0ｍｇ／ｄｌ、ペニシリンＧ 8.0ｍｇ／ｄｌ、フェノ
ールレッド 0.001％、ＥＤＴＡ・２Ｎａ 3.7mg/dlおよ
びＢＳＡ 300ｍｇ／ｄｌを含む）中に入れ、上記導入遺
伝子を受精卵の前核に注入後、偽妊娠したＩＣＲの卵管
に移植した。tau-wild/MoPrPを導入したものからは、37
匹、tau-T837G/MoPrPを導入したものからは54匹のマウ
スが生まれ、ＰＣＲ解析により、導入遺伝子（トランス
ジーン）の有無を確認した結果、それぞれ２匹に導入遺
伝子の挿入を認めた。A cross group was prepared by mating C57BL / 6 with SJL F2. Fertilized eggs obtained from the crosses of these cross groups were modified with Whitten culture solution (NaCl 514 mg
/ Dl, KCl 36.0 mg / dl, KH 2 PO 4 16.0 m
g / dl, MgSO 4 · 7H 2 O 29.0mg / dl, Na
HCO 3 190.0 mg / dl, glucose 100.0 mg / d
1, sodium pyruvate 3.5 mg / dl, calcium lactate / 5H 2 O 53.0 mg / dl, streptomycin
5.0 mg / dl, penicillin G 8.0 mg / dl, phenol red 0.001%, EDTA.2Na 3.7 mg / dl and BSA 300 mg / dl), and the above transgene was injected into the pronucleus of the fertilized egg, and then pseudo Transplanted into the oviduct of a pregnant ICR. From those that introduced tau-wild / MoPrP, 37
Fifty-four mice were born from the transgenic mouse, which had tau-T837G / MoPrP introduced, and the presence or absence of the transgene (transgene) was confirmed by PCR analysis. As a result, insertion of the transgene was confirmed in each of the two mice.
【００２９】６．ＰＣＲによる、マウス中のヒトタウ遺
伝子導入の有無の解析
マウスの尾３ｍｍより、東洋紡績社製MFX-2000を用いて
ゲノムＤＮＡを精製した。得たＤＮＡをテンプレートに
２つのプライマー：
（１）ＤＡ８４:5'-TCCAAGATCGGCTCCACTGA（配列番号１
４）、及び（２）ＤＡ１２３:5'-GTGGATACCCCCTCCCCCAG
CCTAGACC（配列番号１５）を用いてＰＣＲを行った。ヒ
トタウ遺伝子が導入されてるマウスからのサンプルで
は、約650ｂｐのところにバンドが認められるが、ヒト
タウ遺伝子が導入遺伝子されていないマウスからのもの
では、このバンドは認められない。この違いに基づい
て、ヒトタウ遺伝子の導入の有無の確認を行った。6. Analysis of presence / absence of introduction of human tau gene into mouse by PCR Genomic DNA was purified from 3 mm of tail of mouse using MFX-2000 manufactured by Toyobo. Two primers were prepared using the obtained DNA as a template: (1) DA84: 5'-TCCAAGATCGGCTCCACTGA (SEQ ID NO: 1)
4), and (2) DA123: 5'-GTGGATACCCCCTCCCCCAG
PCR was performed using CCTAGACC (SEQ ID NO: 15). In a sample from a mouse into which the human tau gene has been introduced, a band is observed at about 650 bp, but in a mouse from which the human tau gene has not been introduced, this band is not observed. Based on this difference, it was confirmed whether or not the human tau gene had been introduced.
【００３０】得られたヒトタウ遺伝子を発現しているマ
ウス（ファウンダーマウス）をC57BL/6マウスと交配さ
せることによって、トランスジェニックマウスのライン
を作成した。すなわち、ファウンダーマウスを一旦正常
マウスと交配させることにより、F1世代として、トラン
スジーンを相同染色体の片方のみに有するヘテロザイゴ
ート（ヘテロ接合体）マウス及びトランスジーンを有し
ない正常マウスが略１：１の比率で得られる。F1マウス
からサンプル組織を採取して前述にようにしてＰＣＲで
トランスジーンの有無を確認することにより、F1マウス
を、トランスジーンに関してヘテロザイゴートと正常マ
ウスとに区別することができる。次いで、ヘテロザイゴ
ートF1マウス同士を交配させることにより、F2世代マウ
スを作成した。F2マウスは、略４分の１がトランスジー
ンに関しホモザイゴート（ホモ接合体）で有したもの、
４分の２がヘテロザイゴート、残り４分の１がトランス
ジーンを有しないものとなった。ホモザイゴートとヘテ
ロザイゴートとの識別は、F2を正常マウスと交配させて
得るマウス（F3）におけるトランスジーンの有無に基づ
いて行った。すなわち、F3マウスがトランスジーンに関
して全てポジティブであったとき、それらの親であるF2
マウスはトランスジーンに関してホモザイゴートであ
り、F3の中にPCRでトランスジーン陰性のものが含まれ
ている場合は、それらの親であるF2マウスはトランスジ
ーンに関してヘテロザイゴートである。こうしてホモザ
イゴートマウスを確立し、その維持はホモザイゴート同
士の交配により行い、ヘテロザイゴートの作成は、ホモ
ザイゴートと正常マウスとの交配により行った。A line of transgenic mice was prepared by crossing the obtained mice expressing the human tau gene (founder mice) with C57BL / 6 mice. That is, by crossing a founder mouse with a normal mouse once, as an F1 generation, a heterozygous (heterozygous) mouse having a transgene only on one side of a homologous chromosome and a normal mouse having no transgene have a ratio of about 1: 1. Obtained in ratio. By collecting a sample tissue from the F1 mouse and confirming the presence or absence of the transgene by PCR as described above, the F1 mouse can be distinguished as a heterozygote and a normal mouse with respect to the transgene. Next, heterozygous F1 mice were crossed with each other to prepare F2 generation mice. About 1/4 of the F2 mice had homozygotes (homozygotes) for the transgene,
Two-quarters were heterozygous and the remaining one-quarter had no transgene. Discrimination between homozygotes and heterozygotes was performed based on the presence or absence of transgene in the mouse (F3) obtained by mating F2 with a normal mouse. That is, when F3 mice were all positive for the transgene, their parental F2
Mice are homozygous for the transgene, and if their F3 contains a PCR-negative transgene, their parental F2 mice are heterozygous for the transgene. In this way, homozygous mice were established and maintained by mating homozygous with each other, and heterozygotes were created by mating homozygous with normal mice.
【００３１】７．マウス中のヒトタウ遺伝子の発現及び
リン酸化亢進の確認
ヒトタウ遺伝子（野生型／変異型）導入マウスのうち、
トランスジーンをヘテロに有するものを用いた。これら
のマウス（12月齢、各３匹）の分割脳サンプルおよび、
非トランスジェニックマウス（ヒトタウ遺伝子導入操作
を行って作ったマウスであるが、PCR検査、ウエスタン
ブロット検査でヒトタウの発現を認めなかったもの。）
（12月齢、３匹）の全脳サンプルを、サンプルの10倍重
量の細胞溶解緩衝液（50ｍＭ Tris, pH 8.0, 10mM ＥＤ
ＴＡ, 100mM ＮａＣｌ, ２% Triton, 10 ｍＭ 二リン酸
ナトリウム10水和物、20 mM ＮａＦ, 2mM バナジン酸ナ
トリウム、Complete Protease Inhibitor (Boehringer
Mannheim)）でホモジナイズ後、タンパク質濃度を測定
した。得られたライセート10μｇを10％（ｗ／ｖ）ポリ
アクリルアミドゲルで電気泳動後、ウエスタンブロット
解析を行った。7. Confirmation of human tau gene expression and phosphorylation enhancement in mice Among human tau gene (wild type / mutant type) introduced mice,
A heterogeneous transgene was used. Divided brain samples of these mice (12 months old, 3 each), and
Non-transgenic mouse (human Tau gene transfer operation, but no expression of human tau was observed by PCR or Western blot)
A whole brain sample (3 months old, 12 months old) was prepared by adding 10 times the weight of the sample to a cell lysis buffer (50 mM Tris, pH 8.0, 10 mM ED).
TA, 100 mM NaCl, 2% Triton, 10 mM sodium diphosphate decahydrate, 20 mM NaF, 2 mM sodium vanadate, Complete Protease Inhibitor (Boehringer
Protein concentration was measured after homogenization with Mannheim). 10 μg of the obtained lysate was electrophoresed on a 10% (w / v) polyacrylamide gel and then subjected to Western blot analysis.
【００３２】ウエスタンブロット解析で使用した１次抗
体は、HT7 (Innogenitics；マウスタウには反応せず、
ヒトタウを特異的に認識する。)、AT8（Innogenitics；
タウ蛋白の202/205位のリン酸化セリンを含む部分を認
識する。）、AT180（Innogenitics；タウ蛋白の231位の
リン酸化スレオニンを含む部分を認識する。）、AT270
（Innogenitics；タウ蛋白の181位のリン酸化スレオニ
ンを含む部分を認識する。）である。The primary antibody used in Western blot analysis did not react with HT7 (Innogenitics; mouse tau,
Recognizes human tau specifically. ), AT8 (Innogenitics;
Recognize the part containing phosphorylated serine at the 202/205 position of tau protein. ), AT180 (Innogenitics; recognizes a portion containing phosphorylated threonine at position 231 of tau protein), AT270
(Innogenitics; recognizes the portion containing phosphorylated threonine at position 181 of tau protein.).
【００３３】結果：
（１）一次抗体HT7による結果： ヒトタウ遺伝子（野
生型／変異型）導入マウスにおいてのみ、ヒトタウタン
パク質の発現が認められた。また、野生型と変異型でほ
ぼ発現部位、発現量とも同じ様式であった（図８）。
（２）一次抗体AT8, AT180又はAT270による結果： こ
れらの抗体が認めるタウ蛋白のリン酸化は、ヒトタウ遺
伝子（野生型／変異型）導入マウスにおいてのみ認めら
れ、非トランスジェニックマウスには認められなかっ
た。また、リン酸化の程度は、野生型ヒトタウ遺伝子導
入マウスより、変異型遺伝子導入マウスにおける方が強
かった（図９）。Results: (1) Results with primary antibody HT7: Expression of human tau protein was observed only in mice into which human tau gene (wild type / mutant type) was introduced. In addition, the expression type and expression level were almost the same in the wild type and the mutant type (Fig. 8). (2) Results with primary antibody AT8, AT180 or AT270: Phosphorylation of tau protein observed by these antibodies was observed only in the human tau gene (wild type / mutant type) -introduced mouse, not in non-transgenic mouse. It was The degree of phosphorylation was stronger in the mutant transgenic mouse than in the wild-type human tau transgenic mouse (FIG. 9).
【００３４】８．ヒトタウ遺伝子導入マウスの学習能の
低下の測定
野生型（WILD-D）、変異型 (N279K) および、コントロ
ールとして、非トランスジェニックマウス（前記）を試
験に用いた。詳細を次の表に示す。8. Measurement of decrease in learning ability of human tau transgenic mouse Wild type (WILD-D), mutant type (N279K), and non-transgenic mouse (above) as a control were used in the test. Details are shown in the following table.
【００３５】[0035]
【表１】 [Table 1]
【００３６】（１）オープンフィールドテスト
側面を透明なアクリル板で囲った正方形（30×30×30ｃ
ｍ）のオープンフィールド中にマウスをおき、そこにお
けるマウスの水平方向の動き（歩行活動）を、床上２ｃ
ｍにセットした４本の赤外線ビームによって検出した。
ＸとＹのそれぞれの軸に10ｃｍ間隔で配置した２本のビ
ームはフリップフロップ回路を構成し、同じビームへの
繰り返しの反応は記録されないようにした。垂直方向へ
の動き（立ち上がり）は床上５ｃｍに並べられた12個の
赤外線ビームによって検出した。その他の動きを含む総
ての活動は、オープンフィールドの30ｃｍ上方に取り付
けた赤外線の散乱を利用した領域センサー（F5B-GA18M,
オムロン製）により検出した。それぞれのビームをカ
ットした回数はプログラマブルコントローラー（C40, O
MRON）によりインターフェイスされた後、パソコンに記
録された。このオープンフィールド検査は防音し、照明
(40Ｗ)した箱の中で、５分間毎に区切られた連続４ブロ
ックより構成される２０分間にわたって行われ、各時間
ブロック毎に平均ビームカット数（ビームを横切った回
数の平均値）を集計した。(1) Open field test A square (30 × 30 × 30c) surrounded by a transparent acrylic plate on the side.
Place the mouse in the open field of m), and move the mouse horizontally (walking activity) there on the floor 2c.
It was detected by four infrared beams set to m.
Two beams arranged at 10 cm intervals on each of the X and Y axes constituted a flip-flop circuit so that repeated responses to the same beam were not recorded. The vertical movement (rise) was detected by 12 infrared beams arranged 5 cm above the floor. All activities, including other movements, were carried out 30 cm above the open field using infrared scattering area sensors (F5B-GA18M,
OMRON). Programmable controller (C40, O
It was recorded on a personal computer after being interfaced by MRON). This open field inspection is soundproof and lighting
In a box (40W), it was performed for 20 minutes consisting of 4 consecutive blocks divided every 5 minutes, and the average number of beam cuts (average number of times the beam was crossed) was calculated for each time block. did.
【００３７】結果を図１０に示す。図に見られるよう
に、非トランスジェニックマウスと野生型ヒトタウ遺伝
子導入マウスでは、時間経過と共に平均ビームカット数
が減少し、第３時間ブロック（試験開始後10〜15分）な
いし第４ブロック（試験開始後15〜２０分）でプラトー
に達した。これに対し、変異型ヒトタウ遺伝子（N279
K）導入マウスでは、時間経過による平均ビームカット
数の減少は見られなかった。このことは、変異型ヒトタ
ウ遺伝子導入マウスにおいて、環境変化に対する「慣
れ」の能力が低下していることを示している。The results are shown in FIG. As shown in the figure, in non-transgenic mice and wild-type human tau transgenic mice, the average number of beam cuts decreased with the lapse of time, and the third block (10 to 15 minutes after the start of the test) to the fourth block (test) A plateau was reached 15-20 minutes after the start). In contrast, the mutant human tau gene (N279
K) Introduced mice showed no decrease in the average number of beam cuts over time. This indicates that the ability of "accustoming" to environmental changes is reduced in the mutant human tau transgenic mouse.
【００３８】（２）聴覚性驚愕反射と驚愕反射のプレパ
ルス抑制（PPI:prepulse inhibition）
装置として、防音室内に置いた透明のアクリル製の箱
（70×70×165ｍｍ）を用いた。箱の左右は音が直接聞
こえるようにアルミの穴あきパネルの形態とした。箱は
底の四隅付近を弾力のあるボールで支え、その床の中央
の外部に加速度センサー（GH313A, GA-245SO, KEYENC
E）を取り付けた。マウスを箱に入れ、マウスのすべて
の動きを床の振動を用いて検出した。センサーからの電
圧出力はローパスフィルター（60Hz, FV-624, NF)を通
過した後、400ＨｚでA/D変換され（MacLab, A/D Instru
ments）、パソコンに記録された(Startle Amplitude)。
聴覚性驚愕刺激として、115dB(C)のホワイトノイズを１
回50ms与えた。視覚性のプレパルスとして、36個の赤色
LEDからの光を用い、それらはマウスを入れた箱の10ｃ
ｍ上に18個ずつ２列に並べ、１回あたり20ミリ秒与え
た。聴覚性のプレパルスとして、85ｄＢ (C)のホワイト
ノイズを１回あたり30ミリ秒与えた。バックグランドの
騒音レベルは75ｄＢ (C) で、バックグランドの明度レ
ベルは0.1 luxであった。 １セッションは76試行から成
り、最初の40試行は驚愕刺激のみを与える慣れ（habitu
ation）試行とし、続く36試行はプレパルスによるテス
ト試行とした。このプレパルスによるテスト試行では、
50、100または200ミリ秒のリードタイムを持つ音PPと光
PPの試行を４試行ずつ、12試行の 驚愕刺激のみのコン
トロール試行を挟みランダムに行った。平均試行間間隔
は25秒であった。刺激の呈示はプログラマブルコントロ
ーラー(C40,オムロン製) により制御された。 驚愕反射
強度として、驚愕刺激の呈示から200ミリ秒以内の最大
振幅を測定した。(2) Auditory startle reflex and startle reflex prepulse inhibition (PPI: prepulse inhibition) As a device, a transparent acrylic box (70 × 70 × 165 mm) placed in a soundproof room was used. The left and right sides of the box are in the form of perforated aluminum panels so that sound can be heard directly. The box is supported by elastic balls near the four corners of the bottom, and an acceleration sensor (GH313A, GA-245SO, KEYENC) is installed outside the center of the floor.
E) installed. The mouse was placed in a box and all movements of the mouse were detected using floor vibration. The voltage output from the sensor is A / D converted at 400Hz after passing through a low pass filter (60Hz, FV-624, NF) (MacLab, A / D Instrument
ments), recorded on a computer (Startle Amplitude).
115 dB (C) white noise as auditory startle stimulus
Giving 50 ms times. 36 reds as visual prepulses
Using the light from the LED, they are 10c in a box containing a mouse
18 pieces on m were arranged in two rows, and each time was given for 20 milliseconds. As an auditory prepulse, 85 dB (C) white noise was given for 30 ms each. The background noise level was 75 dB (C) and the background brightness level was 0.1 lux. One session consists of 76 trials, and the first 40 trials are used to give only startle stimuli (habitu).
ation) trial, and the following 36 trials were prepulse test trials. In this prepulse test trial,
Sound PP and light with a lead time of 50, 100 or 200 ms
Four PP trials were performed at a time, with 12 trials each including a startle stimulus-only control trial. The average intertrial interval was 25 seconds. The stimulus presentation was controlled by a programmable controller (C40, OMRON). As the startle reflex intensity, the maximum amplitude within 200 ms after the startle stimulus was presented was measured.
【００３９】（結果）結果を図１１に示す。驚愕反射に
おいて、N279Kマウスは、他より反射が強いことがわか
る。プレパルス抑制において、光刺激では抑制が見られ
ないが、音刺激では抑制が見られた（図１２）。さらに
50ミリ秒のリードタイムでは、N279Kマウスでは抑制が
見られないのに対し、野生型ヒトタウ遺伝子導入マウ
ス、および、非トランスジェニックマウスでは抑制が見
られていることが判明した。(Results) The results are shown in FIG. It can be seen that in the startle reflex, N279K mice have a stronger reflex than others. In the prepulse suppression, suppression was not seen by the light stimulation, but suppressed by the sound stimulation (Fig. 12). further
At a lead time of 50 ms, it was revealed that suppression was not observed in N279K mice, whereas suppression was observed in wild-type human tau transgenic mice and non-transgenic mice.
【００４０】（３）受動的回避学習
方法： プラスチック製のシャトルボックス（幅20×奥
行10×高20ｃｍ）を改良した装置１台を使用。シャトル
ボックスはプラスチックのクーラーボックスに内蔵し、
ファンによる換気を施した。また，シャトルボックス内
部は直流24Ｖの豆電球によりを照明し，照度は12 luxで
あった。無条件刺激（US）には被験体と直列に250 ｋΩ
の固定抵抗を経由した140Ｖ（交流）（0.5ｍＡ）のスク
ランブル電撃を用いた。また，刺激の制御と反応の記録
はオムロン社C-40型コントローラを内蔵したシステムに
より行われた。
１）条件づけ試行： 動物をケージに入れたまま飼育室
から装置の間近まで運び、明るい部屋に入れた。仕切り
板を上に上げて試行を開始し、マウスが隣の暗い部屋に
入るまでの時間（条件づけ前の潜時）を自動計測した。
マウスが暗い部屋に入れば仕切り板を下ろし、10秒後に
電気ショックを床から３秒間与えた。
２）20分後のテスト試行： 条件づけを行なわなかった
部屋（明るい部屋）にマウス入れ、条件づけでショック
を受けた部屋（暗い部屋）にマウスが入るまでの時間
（条件づけ後の潜時）を測定した。180秒経過してもマ
ウスが暗い部屋に入らなかったときは、その時点で試行
を終了し、潜時を180秒とみなした。
３）24時間後の再テスト： 上記20分後のテスト試行と
同じ方法で行った。(3) Passive avoidance learning method: A single device with an improved plastic shuttle box (width 20 x depth 10 x height 20 cm) is used. The shuttle box is built in a plastic cooler box,
Ventilation was performed by a fan. Also, the interior of the shuttle box was illuminated with a miniature bulb of 24V DC, and the illuminance was 12 lux. 250 kΩ in series with the subject for unconditional stimulation (US)
The scramble electric shock of 140V (AC) (0.5mA) via the fixed resistance of was used. The control of the stimulus and the recording of the reaction were performed by a system with a built-in OMRON C-40 controller. 1) Conditioning trial: The animal was carried in the cage from the breeding room to the vicinity of the device and placed in a bright room. The partition plate was raised and the trial was started, and the time until the mouse entered the next dark room (latency before conditioning) was automatically measured.
When the mouse entered the dark room, the partition plate was lowered, and after 10 seconds, an electric shock was applied from the floor for 3 seconds. 2) Test trial after 20 minutes: The time until the mouse enters the room that was not conditioned (bright room) and was shocked by the condition (dark room) (latency after conditioning) ) Was measured. If the mouse did not enter the dark room after 180 seconds, the trial was terminated at that point and the latency was considered to be 180 seconds. 3) Retest after 24 hours: The test was performed in the same manner as the test trial after 20 minutes.
【００４１】（結果）結果を図１３に示す。潜時延長が
80秒以下の場合を回避能力が低下したものとして、各動
物の受動的回避学習能力を判別した。その結果、非トラ
ンスジェニックマウスには回避能力の低下は認められな
かったが、トランスジェニックマウスでは50％のマウス
に回避能力の低下が認められた。次いで、回避能力の低
下したマウスについて潜時延長の平均値を求めると、N2
79Kマウスでは、図１４に示すように有意に短縮してお
り、野生型マウスと比べて回避能力の低下の程度が高い
ことが示された。(Results) The results are shown in FIG. Latency extension
The passive avoidance learning ability of each animal was discriminated as the case where the avoidance ability was lowered when the time was 80 seconds or less. As a result, the non-transgenic mice did not show a decrease in evasion ability, but 50% of the transgenic mice showed a decrease in evasion ability. Next, when the average value of the latency prolongation was calculated for the mouse with reduced avoidance ability, N2
As shown in FIG. 14, the 79K mouse had a significantly shortened length, indicating that the degree of avoidance was lower than that of the wild-type mouse.
【００４２】（４）Morris水迷路学習
空間的記憶を評価するため（Morris, R. G. et al., Pl
ace navigation impaired in rats with hippocampal l
esions. Nature 297, 681-3. (1982)）、海馬依存性の
課題の一つであるMorris水迷路試行を行った。直径96ｃ
ｍのプールを用い、無毒性のインディアインクを添加し
て水を不透明にした。水温は25℃に維持した。直径10ｃ
ｍのプラットフォームをプール中の一定位置に固定し
た。プラットフォームは、迷路訓練に際しては水面より
５ｍｍ沈め、明視条件下の訓練に際しては水面上に出現
させた。１日５回の試行による迷路訓練が５回行われ、
続いて、５回の試行による明視プラットフォームテスト
が翌日に行われた。迷路学習及び明視下の試行におい
て、マウスを、プールの４分円のうちプラットフォーム
の設置されていないの３つの四分円の何れかに入れた。
実験者は、マウスが泳いでプラットフォームに到達する
までの時間（潜時）をストップウォッチで計測した。マ
ウスがプラットフォームに到達すると、マウスをその場
に10秒間留め、次いで待機用ボックスに戻した。マウス
がプラットフォームに60秒以内に到達できなかった場合
は、実験者がマウスをプラットフォーム上に乗せ、潜時
を60秒と記録した。試行間の間隔は30秒とした。(4) Morris Water Maze Learning To evaluate spatial memory (Morris, RG et al., Pl
ace navigation impaired in rats with hippocampal l
esions. Nature 297, 681-3. (1982)), a Morris water maze trial, which is one of the problems of hippocampal dependence, was performed. Diameter 96c
The non-toxic India ink was added to make the water opaque using a pool of m. The water temperature was maintained at 25 ° C. Diameter 10c
The m platform was fixed in position in the pool. The platform was submerged by 5 mm from the water surface during the maze training, and was made to appear on the water surface during the training under the visual condition. Maze training is conducted 5 times a day, 5 times a day,
Subsequently, a clear vision platform test with 5 trials was conducted the next day. In the labyrinth learning and visual trials, mice were placed in any of the three non-platform quadrants of the pool quadrant.
The experimenter measured the time (latency) for the mouse to swim and reach the platform with a stopwatch. When the mouse reached the platform, it was held in place for 10 seconds and then returned to the waiting box. If the mouse was unable to reach the platform within 60 seconds, the experimenter placed the mouse on the platform and recorded a latency of 60 seconds. The interval between trials was 30 seconds.
【００４３】（結果）隠れたプラットフォームの位置を
見つけるために、変異型ヒトタウ発現（N279K）マウス
は、野生型ヒトタウ発現マウス及び非トランスジェニッ
クマウスに比して、有意に長い潜時を必要とした（図１
５）。野型マウスと非トランスジェニックマウスとの間
には、有意差はなかった。結果は、N279Kマウスにおけ
る水泳速度の低下によるものではなく、経路の長さが長
くなることによるものであった（データ示さず）。明視
下では、N269Kマウスも野生型マウスも同じように学習
しており、このことは、両者ともにプラットフォームを
見つけることができたことを示している。従って、N279
Kマウスにおいて何らかの視覚機能障害があったとする
理由もない。これらのデータは、N279Kマウスが、空間
的記憶の中枢である海馬に、何らかの機能障害を有する
ことを示唆している。(Results) In order to locate the hidden platform, mutant human tau-expressing (N279K) mice required a significantly longer latency than wild-type human tau-expressing mice and non-transgenic mice. (Fig. 1
5). There was no significant difference between wild type and non-transgenic mice. The results were not due to decreased swimming speed in N279K mice, but due to increased path length (data not shown). Under clear vision, both N269K and wild-type mice were learning the same, indicating that both were able to find the platform. Therefore, N279
There is no reason to say that there was any visual dysfunction in K mice. These data suggest that N279K mice have some dysfunction in the hippocampus, which is the center of spatial memory.
【００４４】（５）能動的回避学習
アクリル製のシャトルボックス（幅20×奥行10×高さ20
ｃｍ）を使用した。条件付け刺激（CS）はトーン（1500
Hz、85ｄB（ｃ））であり、無条件刺激（US）は、マウ
スのいる格子状の床にかけられるスクランブル電撃（交
流160V、0.5ｍＡ）とした。１日50回の試行を３日間行
うことにより、マウスを訓練した。試行は、条件付け刺
激を与え、その後に無条件刺激を与えることによって開
始した。CS-US間隔は５秒とした。試行間の平均間隔は2
5秒とした（ランダムに、10秒〜40秒）。(5) Active avoidance learning Acrylic shuttle box (width 20 x depth 10 x height 20
cm) was used. Conditioning stimulus (CS) is a tone (1500
Hz, 85 dB (c)), and the unconditional stimulation (US) was a scramble electric shock (160 V AC, 0.5 mA) applied to the lattice floor with mice. Mice were trained by performing 50 trials per day for 3 days. Trials were initiated by applying a conditioned stimulus followed by an unconditioned stimulus. The CS-US interval was 5 seconds. Average interval between trials is 2
It was set to 5 seconds (randomly, 10 to 40 seconds).
【００４５】（結果）２回目の試行で能力を回復したも
のの、変異型ヒトタウ発現マウスは、回避学習の遅延を
示した（図１６）。可能な説明の一つは、変異型ヒトタ
ウ発現マウスが、海馬及び視床下部を含む辺縁系の介在
する何らかの感情的欠陥を有するということである。変
異型ヒトタウ発現マウスにおける運動停止等のような感
情的活性化は、恐ろしいショックを回避するのに必要な
行動の開始を妨げると考えられる。別の可能性として、
それらのマウスが、恐怖を記憶することにおいて何らか
の問題を有するということがある。これまでの多くの証
拠が、扁桃体、及び特に外側扁桃体が、恐怖条件の記憶
の貯蔵の基礎をなす可塑性の部位であるらしいことを示
唆している（Schafe, G. E. et al. Memory consolidat
ion of Pavlovianfear conditioning: a cellular and
molecular perspective. Trends Neurosci 24, 540-6.
(2001)）。生後８月齢までの変異型ヒトタウ発現マウス
において、光学顕微鏡的には、扁桃体における明らかな
神経細胞の損失は認められなかったが、回避学習の結果
は、剖検において扁桃体の神経細胞の重大な損失を示す
N279K変異を有する多くの患者と一致している（Yasuda,
M. et al. A mutation inthe microtubule-associated
protein tau in pallido-nigro-luysian degeneratio
n. Neurology 53, 864-8. (1999).）。扁桃体における
疾患の非常に進んだ段階において、神経細胞の損失が起
こるためであろう。(Results) Although the ability was restored in the second trial, the mutant human tau-expressing mouse showed a delay in avoidance learning (FIG. 16). One possible explanation is that mutant human tau-expressing mice have some emotional defects mediated by the limbic system, including the hippocampus and hypothalamus. Emotional activation, such as locomotor arrest, in mutant human tau-expressing mice is thought to interfere with the initiation of behaviors necessary to avoid dreadful shock. Another possibility is
It is possible that the mice have some problems in remembering fear. Much evidence to date suggests that the amygdala, and especially the lateral amygdala, appear to be the site of plasticity underlying memory storage of fear conditions (Schafe, GE et al. Memory consolidat).
ion of Pavlovianfear conditioning: a cellular and
molecular perspective. Trends Neurosci 24, 540-6.
(2001)). In the mutant human tau-expressing mice up to 8 months of age, no apparent neuron loss in the amygdala was observed by light microscopy, but the result of avoidance learning was that a significant loss of amygdala neurons was observed at autopsy. Show
Consistent with many patients with the N279K mutation (Yasuda,
M. et al. A mutation in the microtubule-associated
protein tau in pallido-nigro-luysian degeneratio
n. Neurology 53, 864-8. (1999).). This may be because neuronal loss occurs at a very advanced stage of the disease in the amygdala.
【００４６】（６）電気生理学的解析
空間的な学習及び記憶は、海馬の統合的制御機能を必要
とすることが知られており（Bliss, T. V. & Collingri
dge, G. L. A synaptic model of memory: long-term p
otentiation in the hippocampus. Nature 361, 31-9.
(1993).）、また長期増強は、シナプス伝達の効率の長
期間続く増大（Bliss, T. V. et al., T. Long-lasting
potentiation of synaptic transmission in the dent
ate areaof the anaesthetized rabbit following stim
ulation of the perforant path. J Physiol 232, 331-
56. (1973)）として、学習及び記憶に関係した可塑性変
化の基礎にあるものとされている（Bliss, T. V. et a
l., A synaptic model ofmemory: long-term potentiat
ion in the hippocampus. Nature 361, 31-9. (1993)；
Doyere, V. et al., Linear relationship between the
maintenance ofhippocampal long-term potentiation
and retention of an associative memory. Hippocampu
s 2, 39-48. (1992)）。そこで、マウスの歯状回におけ
るin vivo長期増強（long-term potentiation）の記録
を行った。(6) Electrophysiological analysis It is known that spatial learning and memory require an integrated control function of the hippocampus (Bliss, TV & Collingri).
dge, GL A synaptic model of memory: long-term p
otentiation in the hippocampus. Nature 361, 31-9.
(1993).), And long-term potentiation also increased the efficiency of synaptic transmission over a long period (Bliss, TV et al., T. Long-lasting.
potentiation of synaptic transmission in the dent
ate areaof the anaesthetized rabbit following stim
ulation of the perforant path. J Physiol 232, 331-
56. (1973)) as the basis for plasticity changes related to learning and memory (Bliss, TV et a.
l., A synaptic model of memory: long-term potentiat
ion in the hippocampus. Nature 361, 31-9. (1993);
Doyere, V. et al., Linear relationship between the
maintenance of hippocampal long-term potentiation
and retention of an associative memory. Hippocampu
s 2, 39-48. (1992)). Therefore, we recorded in vivo long-term potentiation in the dentate gyrus of mice.
【００４７】すなわち、マウス〔ヒト変異型タウ発現マ
ウス（７匹）、野生型ヒトタウ発現マウス（７匹）、及
び非トランスジェニックマウス（７匹）〕をウレタン麻
酔（1.2ｇ／ｋｇ、腹腔、及びその後必要に応じて0.2〜
0.6ｇ／ｋｇ、腹腔）し、定位装置に固定した。加熱マ
ットを用いて体温を37℃に維持した。0.9％食塩水を満
たした９〜12μｍの先端径を有するガラス製の記録電極
を、歯状回の細胞体層へと下ろした。最初の応答が、貫
通路の陰極刺激（6.0〜8.0Ｖ、0.1Ｈｚ、0.1ｍｓ持続）
を用いて得られた。電極の挿入及び細胞集団の応答を得
た後、ベースラインを記録するために60分間、系を安定
化させた。ベースラインのスパイク強度が最大漸近値の
１／３となるように電圧を低下させた。用いた長期増強
誘導電圧は、最大漸近スパイク強度を惹起することので
きる最低の電圧レベルとした。長期増強を誘導するため
の持続性硬直のパラメーター（HFS：high frequency st
imulation）は、８回にわたる0.4ミリ秒の400Hzパルス
よりなった。マウスに与える障害を最小限に止めるた
め、長期増強に際したEPSP（興奮性シナプス後電位）勾
配なしに、細胞集団のスパイクのみプロットした。これ
は、この操作では、細胞集団のスパイクの増強変化はEP
SP勾配のそれと類似しているからである（Namgung, U.,
Valcourt, E. & Routtenberg, A. Long-term potentia
tion in vivo in the intact mouse hippocampus. Brai
n Res 689, 85-92. (1995)）。細胞集団スパイクの強度
は、最初のプラスの値からマイナスのピークの値まで測
定した。５分間隔で、５回の連続的刺激によって誘導さ
れた細胞集団スパイクを平均し、解析した。That is, mice [human mutant tau-expressing mice (7 mice), wild-type human tau-expressing mice (7 mice), and non-transgenic mice (7 mice)] were anesthetized with urethane (1.2 g / kg, abdominal cavity, and Then 0.2 ~ as needed
0.6 g / kg, abdominal cavity) and fixed on a stereotaxic apparatus. Body temperature was maintained at 37 ° C using a heating mat. A glass recording electrode with a tip diameter of 9-12 μm filled with 0.9% saline was lowered into the cell body layer of the dentate gyrus. The first response is cathodic stimulation of the through passage (6.0-8.0V, 0.1Hz, 0.1ms duration)
Was obtained using. After obtaining electrode insertion and cell population response, the system was allowed to stabilize for 60 minutes to record a baseline. The voltage was lowered so that the baseline spike intensity was 1/3 of the maximum asymptotic value. The long-term enhanced induction voltage used was the lowest voltage level that could induce the maximum asymptotic spike strength. Parameters of persistent rigidity (HFS: high frequency st) to induce long-term potentiation
imulation) consisted of 0.4 millisecond 400 Hz pulses over eight times. To minimize damage to mice, only spikes in the cell population were plotted, without EPSP (excitatory postsynaptic potential) gradient during long-term potentiation. This is because in this procedure, the enhanced changes in spikes in the cell population are EP
Because it is similar to that of the SP gradient (Namgung, U.,
Valcourt, E. & Routtenberg, A. Long-term potentia
tion in vivo in the intact mouse hippocampus. Brai
n Res 689, 85-92. (1995)). The intensity of the cell population spike was measured from the first positive value to the negative peak value. Cell population spikes induced by 5 consecutive stimuli at 5 minute intervals were averaged and analyzed.
【００４８】（結果）図１７に、持続性強直の前後にお
ける、変異型ヒトタウ発現（N279K）マウス、野生型ヒ
トタウ発現マウス及び非トランスジェニック（非Tg）マ
ウスの顆粒細胞から記録された代表的トレースを示す。
図１８に、変異型マウス（黒丸）、野生型マウス（黒三
角）及び非トランスジェニックマウス（白四角）におけ
る長期増強の推移を、０分における持続性筋強直後から
120分にわたって示す。各点は、平均±標準誤差を、０
分における集団のベースラインスパイク強度に対するパ
ーセントで示す。図１９に、持続性筋強直後の120にわ
たる細胞集団のスパイクの平均±標準誤差を示す（*: p
<0.05, **: p<0.01）。これらの図は、トランスジェニ
ックマウスは、非トランスジェニックマウスに比して、
長期増強が有意に障害されていること、更には、変異型
ヒトタウ発現マウスは、野生型ヒトタウ発現マウスより
もさらに障害されていることを明らかにしている(Results) FIG. 17 shows representative traces recorded from granule cells of mutant human tau-expressing (N279K) mice, wild-type human tau-expressing mice and non-transgenic (non-Tg) mice before and after persistent ankylosis. Indicates.
FIG. 18 shows changes in long-term potentiation in mutant mice (black circles), wild-type mice (black triangles) and non-transgenic mice (white squares) immediately after continuous muscle strength at 0 minutes.
Shown over 120 minutes. Each point has a mean ± standard error of 0
Shown as a percentage of the baseline spike intensity of the population in minutes. FIG. 19 shows the mean ± standard error of the spikes of the cell population over 120 immediately after continuous muscle strengthening (*: p
<0.05, **: p <0.01). These figures show that transgenic mice, compared to non-transgenic mice,
Long-term potentiation is significantly impaired, and moreover, mutant human tau-expressing mice are more impaired than wild-type human tau-expressing mice.
【００４９】[0049]
【発明の効果】以上の一連の検討から、１）ヒトタウ蛋
白を発現するマウスでは、痴呆患者の脳で見られると同
様なタウ蛋白のリン酸化の亢進が認められること、２）
リン酸化の程度は、変異型タウを発現しているマウスの
方が野生型タウを発現しているマウスよりも強い、３）
ヒトタウ蛋白を発現するマウスでは、学習能（受動的及
び能動的回避能力、水迷路学習）の低下、及び記憶及び
学習に関係したシナプス可塑性の基礎にある長期増強の
障害を認めること、及び４）それら学習能の低下や長期
増強の障害の程度は、変異型タウを発現しているマウス
の方が野生型タウを発現しているマウスよりも強いこ
と、が明らかとなった。従って、本発明の変異型ヒトタ
ウ遺伝子を導入したトランスジェニックマウスは、アツ
ルハイマー病、FTDP-17、ピック病、進行性核上性麻
痺、大脳皮質基底核神経節変成症等、タウタンパク質の
異常が関与するタイプの痴呆のモデルマウスとして、病
態発症のメカニズムの解明に、及び抗痴呆薬を開発する
ための各種薬物のスクリーニングのために用いることが
できる。EFFECTS OF THE INVENTION From the above series of investigations, 1) that in mice expressing human tau protein, the same phosphorylation of tau protein as observed in the brain of dementia patients is observed, 2).
The degree of phosphorylation is stronger in mice expressing mutant tau than in mice expressing wild-type tau 3).
In mice expressing the human tau protein, reduced learning ability (passive and active avoidance ability, water maze learning) and impairment of long-term potentiation underlying memory and learning-related synaptic plasticity, and 4) It was revealed that the degree of impaired learning ability and impaired long-term potentiation was stronger in mice expressing mutant tau than in mice expressing wild-type tau. Therefore, the transgenic mouse into which the mutant human tau gene of the present invention has been introduced is involved in tau protein abnormalities such as Azulheimer's disease, FTDP-17, Pick's disease, progressive supranuclear palsy, cerebral corticobasal ganglion metamorphosis, etc. As a model mouse for dementia of the type described above, it can be used to elucidate the mechanism of pathogenesis and to screen various drugs for developing anti-dementia drugs.
【００５０】[0050]
【配列表】
Sequence Listing
<110> TANIGUCHI, Taizo; KITAMURA, Yoshihisa; MORI, Hiroshi
<120> A Mouse Expressing a Mutant Human Tau Gene
<130> P166-02
<150> JP 2001-226596
<151> 2001-07-26
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Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His
20 25 30
caa gac caa gag ggt gac acg gac gct ggc ctg aaa gct gaa gaa gca 144
Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu Lys Ala Glu Glu Ala
35 40 45
ggc att gga gac acc ccc agc ctg gaa gac gaa gct gct ggt cac gtg 192
Gly Ile Gly Asp Thr Pro Ser Leu Glu Asp Glu Ala Ala Gly His Val
50 55 60
acc caa gct cgc atg gtc agt aaa agc aaa gac ggg act gga agc gat 240
Thr Gln Ala Arg Met Val Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp
65 70 75 80
gac aaa aaa gcc aag ggg gct gat ggt aaa acg aag atc gcc aca ccg 288
Asp Lys Lys Ala Lys Gly Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro
85 90 95
cgg gga gca gcc cct cca ggc cag aag ggc cag gcc aac gcc acc agg 336
Arg Gly Ala Ala Pro Pro Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg
100 105 110
att cca gca aaa acc ccg ccc gct cca aag aca cca ccc agc tct ggt 384
Ile Pro Ala Lys Thr Pro Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly
115 120 125
gaa cct cca aaa tca ggg gat cgc agc ggc tac agc agc ccc ggc tcc 432
Glu Pro Pro Lys Ser Gly Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser
130 135 140
cca ggc act ccc ggc agc cgc tcc cgc acc ccg tcc ctt cca acc cca 480
Pro Gly Thr Pro Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro
145 150 155 160
ccc acc cgg gag ccc aag aag gtg gca gtg gtc cgt act cca ccc aag 528
Pro Thr Arg Glu Pro Lys Lys Val Ala Val Val Arg Thr Pro Pro Lys
165 170 175
tcg ccg tct tcc gcc aag agc cgc ctg cag aca gcc ccc gtg ccc atg 576
Ser Pro Ser Ser Ala Lys Ser Arg Leu Gln Thr Ala Pro Val Pro Met
180 185 190
cca gac ctg aag aat gtc aag tcc aag atc ggc tcc act gag aac ctg 624
Pro Asp Leu Lys Asn Val Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu
195 200 205
aag cac cag ccg gga ggc ggg aag gtg cag ata att aag aag aag ctg 672
Lys His Gln Pro Gly Gly Gly Lys Val Gln Ile Ile Lys Lys Lys Leu
210 215 220
gat ctt agc aac gtc cag tcc aag tgt ggc tca aag gat aat atc aaa 720
Asp Leu Ser Asn Val Gln Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys
225 230 235 240
cac gtc ccg gga ggc ggc agt gtg caa ata gtc tac aaa cca gtt gac 768
His Val Pro Gly Gly Gly Ser Val Gln Ile Val Tyr Lys Pro Val Asp
245 250 255
ctg agc aag gtg acc tcc aag tgt ggc tca tta ggc aac atc cat cat 816
Leu Ser Lys Val Thr Ser Lys Cys Gly Ser Leu Gly Asn Ile His His
260 265 270
aaa cca gga ggt ggc cag gtg gaa gta aaa tct gag aag ctt gac ttc 864
Lys Pro Gly Gly Gly Gln Val Glu Val Lys Ser Glu Lys Leu Asp Phe
275 280 285
aag gac aga gtc cag tcg aag att ggg tcc ctg gac aat atc acc cac 912
Lys Asp Arg Val Gln Ser Lys Ile Gly Ser Leu Asp Asn Ile Thr His
290 295 300
gtc cct ggc gga gga aat aaa aag att gaa acc cac aag ctg acc ttc 960
Val Pro Gly Gly Gly Asn Lys Lys Ile Glu Thr His Lys Leu Thr Phe
305 310 315 320
cgc gag aac gcc aaa gcc aag aca gac cac ggg gcg gag atc gtg tac 1008
Arg Glu Asn Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile Val Tyr
325 330 335
aag tcg cca gtg gtg tct ggg gac acg tct cca cgg cat ctc agc aat 1056
Lys Ser Pro Val Val Ser Gly Asp Thr Ser Pro Arg His Leu Ser Asn
340 345 350
gtc tcc tcc acc ggc agc atc gac atg gta gac tcg ccc cag ctc gcc 1104
Val Ser Ser Thr Gly Ser Ile Asp Met Val Asp Ser Pro Gln Leu Ala
355 360 365
acg cta gct gac gag gtg tct gcc tcc ctg gcc aag cag ggt ttg tga 1152
Thr Leu Ala Asp Glu Val Ser Ala Ser Leu Ala Lys Gln Gly Leu
370 375 380
<210> 4
<211> 383
<212> PRN
<213> Homo sapiens
<400> 4
Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly
1 5 10 15
Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His
20 25 30
Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu Lys Ala Glu Glu Ala
35 40 45
Gly Ile Gly Asp Thr Pro Ser Leu Glu Asp Glu Ala Ala Gly His Val
50 55 60
Thr Gln Ala Arg Met Val Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp
65 70 75 80
Asp Lys Lys Ala Lys Gly Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro
85 90 95
Arg Gly Ala Ala Pro Pro Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg
100 105 110
Ile Pro Ala Lys Thr Pro Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly
115 120 125
Glu Pro Pro Lys Ser Gly Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser
130 135 140
Pro Gly Thr Pro Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro
145 150 155 160
Pro Thr Arg Glu Pro Lys Lys Val Ala Val Val Arg Thr Pro Pro Lys
165 170 175
Ser Pro Ser Ser Ala Lys Ser Arg Leu Gln Thr Ala Pro Val Pro Met
180 185 190
Pro Asp Leu Lys Asn Val Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu
195 200 205
Lys His Gln Pro Gly Gly Gly Lys Val Gln Ile Ile Lys Lys Lys Leu
210 215 220
Asp Leu Ser Asn Val Gln Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys
225 230 235 240
His Val Pro Gly Gly Gly Ser Val Gln Ile Val Tyr Lys Pro Val Asp
245 250 255
Leu Ser Lys Val Thr Ser Lys Cys Gly Ser Leu Gly Asn Ile His His
260 265 270
Lys Pro Gly Gly Gly Gln Val Glu Val Lys Ser Glu Lys Leu Asp Phe
275 280 285
Lys Asp Arg Val Gln Ser Lys Ile Gly Ser Leu Asp Asn Ile Thr His
290 295 300
Val Pro Gly Gly Gly Asn Lys Lys Ile Glu Thr His Lys Leu Thr Phe
305 310 315 320
Arg Glu Asn Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile Val Tyr
325 330 335
Lys Ser Pro Val Val Ser Gly Asp Thr Ser Pro Arg His Leu Ser Asn
340 345 350
Val Ser Ser Thr Gly Ser Ile Asp Met Val Asp Ser Pro Gln Leu Ala
355 360 365
Thr Leu Ala Asp Glu Val Ser Ala Ser Leu Ala Lys Gln Gly Leu
370 375 380
<210> 5
<211> 1326
<212> DNA
<213> Homo sapiens
<400> 5
atg gct gag ccc cgc cag gag ttc gaa gtg atg gaa gat cac gct ggg 48
Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly
1 5 10 15
acg tac ggg ttg ggg gac agg aaa gat cag ggg ggc tac acc atg cac 96
Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His
20 25 30
caa gac caa gag ggt gac acg gac gct ggc ctg aaa gaa tct ccc ctg 144
Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu Lys Glu Ser Pro Leu
35 40 45
cag acc ccc act gag gac gga tct gag gaa ccg ggc tct gaa acc tct 192
Gln Thr Pro Thr Glu Asp Gly Ser Glu Glu Pro Gly Ser Glu Thr Ser
50 55 60
gat gct aag agc act cca aca gcg gaa gat gtg aca gca ccc tta gtg 240
Asp Ala Lys Ser Thr Pro Thr Ala Glu Asp Val Thr Ala Pro Leu Val
65 70 75 80
gat gag gga gct ccc ggc aag cag gct gcc gcg gag ccc cac acg gag 288
Asp Glu Gly Ala Pro Gly Lys Gln Ala Ala Ala Glu Pro His Thr Glu
85 90 95
atc cca gaa gga acc aca gct gaa gaa gca ggc att gga gac acc ccc 336
Ile Pro Glu Gly Thr Thr Ala Glu Glu Ala Gly Ile Gly Asp Thr Pro
100 105 110
agc ctg gaa gac gaa gct gct ggt cac gtg acc caa gct cgc atg gtc 384
Ser Leu Glu Asp Glu Ala Ala Gly His Val Thr Gln Ala Arg Met Val
115 120 125
agt aaa agc aaa gac ggg act gga agc gat gac aaa aaa gcc aag ggg 432
Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp Asp Lys Lys Ala Lys Gly
130 135 140
gct gat ggt aaa acg aag atc gcc aca ccg cgg gga gca gcc cct cca 480
Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro Arg Gly Ala Ala Pro Pro
145 150 155 160
ggc cag aag ggc cag gcc aac gcc acc agg att cca gca aaa acc ccg 528
Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg Ile Pro Ala Lys Thr Pro
165 170 175
ccc gct cca aag aca cca ccc agc tct ggt gaa cct cca aaa tca ggg 576
Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly Glu Pro Pro Lys Ser Gly
180 185 190
gat cgc agc ggc tac agc agc ccc ggc tcc cca ggc act ccc ggc agc 624
Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser
195 200 205
cgc tcc cgc acc ccg tcc ctt cca acc cca ccc acc cgg gag ccc aag 672
Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys
210 215 220
aag gtg gca gtg gtc cgt act cca ccc aag tcg ccg tct tcc gcc aag 720
Lys Val Ala Val Val Arg Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys
225 230 235 240
agc cgc ctg cag aca gcc ccc gtg ccc atg cca gac ctg aag aat gtc 768
Ser Arg Leu Gln Thr Ala Pro Val Pro Met Pro Asp Leu Lys Asn Val
245 250 255
aag tcc aag atc ggc tcc act gag aac ctg aag cac cag ccg gga ggc 816
Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu Lys His Gln Pro Gly Gly
260 265 270
ggg aag gtg cag ata att aat aag aag ctg gat ctt agc aac gtc cag 864
Gly Lys Val Gln Ile Ile Asn Lys Lys Leu Asp Leu Ser Asn Val Gln
275 280 285
tcc aag tgt ggc tca aag gat aat atc aaa cac gtc ccg gga ggc ggc 912
Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys His Val Pro Gly Gly Gly
290 295 300
agt gtg caa ata gtc tac aaa cca gtt gac ctg agc aag gtg acc tcc 960
Ser Val Gln Ile Val Tyr Lys Pro Val Asp Leu Ser Lys Val Thr Ser
305 310 315 320
aag tgt ggc tca tta ggc aac atc cat cat aaa cca gga ggt ggc cag 1008
Lys Cys Gly Ser Leu Gly Asn Ile His His Lys Pro Gly Gly Gly Gln
325 330 335
gtg gaa gta aaa tct gag aag ctt gac ttc aag gac aga gtc cag tcg 1056
Val Glu Val Lys Ser Glu Lys Leu Asp Phe Lys Asp Arg Val Gln Ser
340 345 350
aag att ggg tcc ctg gac aat atc acc cac gtc cct ggc gga gga aat 1104
Lys Ile Gly Ser Leu Asp Asn Ile Thr His Val Pro Gly Gly Gly Asn
355 360 365
aaa aag att gaa acc cac aag ctg acc ttc cgc gag aac gcc aaa gcc 1152
Lys Lys Ile Glu Thr His Lys Leu Thr Phe Arg Glu Asn Ala Lys Ala
370 375 380
aag aca gac cac ggg gcg gag atc gtg tac aag tcg cca gtg gtg tct 1200
Lys Thr Asp His Gly Ala Glu Ile Val Tyr Lys Ser Pro Val Val Ser
385 390 395 400
ggg gac acg tct cca cgg cat ctc agc aat gtc tcc tcc acc ggc agc 1248
Gly Asp Thr Ser Pro Arg His Leu Ser Asn Val Ser Ser Thr Gly Ser
405 410 415
atc gac atg gta gac tcg ccc cag ctc gcc acg cta gct gac gag gtg 1296
Ile Asp Met Val Asp Ser Pro Gln Leu Ala Thr Leu Ala Asp Glu Val
420 425 430
tct gcc tcc ctg gcc aag cag ggt ttg tga 1326
Ser Ala Ser Leu Ala Lys Gln Gly Leu
435 440
<210> 6
<211> 441
<212> PRN
<213> Homo sapiens
<400> 6
Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly
1 5 10 15
Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His
20 25 30
Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu Lys Glu Ser Pro Leu
35 40 45
Gln Thr Pro Thr Glu Asp Gly Ser Glu Glu Pro Gly Ser Glu Thr Ser
50 55 60
Asp Ala Lys Ser Thr Pro Thr Ala Glu Asp Val Thr Ala Pro Leu Val
65 70 75 80
Asp Glu Gly Ala Pro Gly Lys Gln Ala Ala Ala Glu Pro His Thr Glu
85 90 95
Ile Pro Glu Gly Thr Thr Ala Glu Glu Ala Gly Ile Gly Asp Thr Pro
100 105 110
Ser Leu Glu Asp Glu Ala Ala Gly His Val Thr Gln Ala Arg Met Val
115 120 125
Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp Asp Lys Lys Ala Lys Gly
130 135 140
Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro Arg Gly Ala Ala Pro Pro
145 150 155 160
Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg Ile Pro Ala Lys Thr Pro
165 170 175
Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly Glu Pro Pro Lys Ser Gly
180 185 190
Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser
195 200 205
Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys
210 215 220
Lys Val Ala Val Val Arg Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys
225 230 235 240
Ser Arg Leu Gln Thr Ala Pro Val Pro Met Pro Asp Leu Lys Asn Val
245 250 255
Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu Lys His Gln Pro Gly Gly
260 265 270
Gly Lys Val Gln Ile Ile Asn Lys Lys Leu Asp Leu Ser Asn Val Gln
275 280 285
Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys His Val Pro Gly Gly Gly
290 295 300
Ser Val Gln Ile Val Tyr Lys Pro Val Asp Leu Ser Lys Val Thr Ser
305 310 315 320
Lys Cys Gly Ser Leu Gly Asn Ile His His Lys Pro Gly Gly Gly Gln
325 330 335
Val Glu Val Lys Ser Glu Lys Leu Asp Phe Lys Asp Arg Val Gln Ser
340 345 350
Lys Ile Gly Ser Leu Asp Asn Ile Thr His Val Pro Gly Gly Gly Asn
355 360 365
Lys Lys Ile Glu Thr His Lys Leu Thr Phe Arg Glu Asn Ala Lys Ala
370 375 380
Lys Thr Asp His Gly Ala Glu Ile Val Tyr Lys Ser Pro Val Val Ser
385 390 395 400
Gly Asp Thr Ser Pro Arg His Leu Ser Asn Val Ser Ser Thr Gly Ser
405 410 415
Ile Asp Met Val Asp Ser Pro Gln Leu Ala Thr Leu Ala Asp Glu Val
420 425 430
Ser Ala Ser Leu Ala Lys Gln Gly Leu
435 440
<210> 7
<211> 42
<212> DNA
<213> Homo sapiens
<400> 7
ggcgggaagg tgcagataat taagaagaag ctggatctta gc 42
<210> 8
<211> 42
<212> DNA
<213> Homo sapiens
<400> 8
gctaagatcc agcttcttct taattatctg caccttcccg cc 42
<210> 9
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> A sequence constructed by ligating an XhoI-site sequence to 5' end
of the start codon of human tau cDNA.
<400> 9
gtggatccct cgagcccatg gctgagcccc gccaggagtt 40
<210> 10
<211> 28
<212> DNA
<213> Homo sapiens
<400> 10
gagtcgactt gtcatcgctt ccagtccc 28
<210> 11
<211> 765
<212> DNA
<213> Mus musculus
<400> 11
atggcgaacc ttggctactg gctgctggcc ctctttgtga ctatgtggac tgatgtcggc 60
ctctgcaaaa agcggccaaa gcctggaggg tggaacaccg gtggaagccg gtatcccggg 120
cagggaagcc ctggaggcaa ccgttaccca cctcagggtg gcacctgggg gcagccccac 180
ggtggtggct ggggacaacc ccatgggggc agctggggac aacctcatgg tggtagttgg 240
ggtcagcccc atggcggtgg atggggccaa ggagggggta cccataatca gtggaacaag 300
cccagcaaac caaaaaccaa cttcaagcat gtggcagggg ctgcggcagc tggggcagta 360
gtggggggcc ttggtggcta catgctgggg agcgccatga gcaggcccat gatccatttt 420
ggcaacgact gggaggaccg ctactaccgt gaaaacatgt accgctaccc taaccaagtg 480
tactacaggc cagtggatca gtacagcaac cagaacaact tcgtgcacga ctgcgtcaat 540
atcaccatca agcagcacac ggtcgtcacc accaccaagg gggagaactt caccgagacc 600
gatgtgaaga tgatggagcg cgtggtggag cagatgtgcg tcacccagta ccagaaggag 660
tcccaggcct attacgacgg gagaagatcc agcagcaccg tgcttttctc ctcccctcct 720
gtcatcctcc tcatctcctt cctcatcttc ctgatcgtgg gatga 765
<210> 12
<211> 98
<212> DNA
<213> Mus musculus
<400> 12
gactcctgag tatatttcag aactgaacca tttcaaccga gctgaagcat tctgccttcc 60
tagtggtacc agtccaattt aggagagcca agcagact 98
<210> 13
<211> 29
<212> DNA
<213> Mus musculus
<400> 13
ggggtacctc gagccttcct gcttgttcc 29
<210> 14
<211> 20
<212> DNA
<213> Homo sapiens
<400> 14
tccaagatcg gctccactga 20
<210> 15
<211> 28
<212> DNA
<213> Homo sapiens
<400> 15
gtggataccc cctcccccag cctagacc ２
８[Sequence Listing] Sequence Listing <110> TANIGUCHI, Taizo; KITAMURA, Yoshihisa; MORI, Hiroshi <120> A Mouse Expressing a Mutant Human Tau Gene <130> P166-02 <150> JP 2001-226596 <151> 2001-07 -26 <160><210> 1 <211> 1152 <212> DNA <213> Homo sapiens <400> 1 atg gct gag ccc cgc cag gag ttc gaa gtg atg gaa gat cac gct ggg 48 Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly 1 5 10 15 acg tac ggg ttg ggg gac agg aaa gat cag ggg ggc tac acc atg cac 96 Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His 20 25 30 caa gac caa gag ggt gac acg gac gct ggc ctg aaa gct gaa gaa gca 144 Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu Lys Ala Glu Glu Ala 35 40 45 ggc att gga gac acc ccc agc ctg gaa gac gaa gct gct gct gtg 192 Gly Ile Gly Asp Thr Pro Ser Leu Glu Asp Glu Ala Ala Gly His Val 50 55 60 acc caa gct cgc atg gtc agt aaa agc aaa gac ggg act gga agc gat 240 Thr Gln Ala Arg Met Val Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp 65 70 75 80 gac aaa aaa gcc aag ggg gct gat ggt aa a acg aag atc gcc aca ccg 288 Asp Lys Lys Ala Lys Gly Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro 85 90 95 cgg gga gca gcc cct cca ggc cag aag ggc cag gcc aac gcc acc agg 336 Arg Gly Ala Ala Pro Pro Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg 100 105 110 att cca gca aaa acc ccg ccc gct cca aag aca cca ccc agc tct ggt 384 Ile Pro Ala Lys Thr Pro Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly 115 120 125 gaa cct cca aaa tca ggg gat cgc agc ggc tac agc agc ccc ggc tcc 432 Glu Pro Pro Lys Ser Gly Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser 130 135 140 cca ggc act ccc ggc agc cgc tcc cgc acc ccg tcc ctt ctt cca 480 Pro Gly Thr Pro Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro 145 150 155 160 ccc acc cgg gag ccc aag aag gtg gca gtg gtc cgt act cca ccc aag 528 Pro Thr Arg Glu Pro Lys Lys Val Ala Val Val Arg Thr Pro Pro Lys 165 170 175 tcg ccg tct tcc gcc aag agc cgc ctg cag aca gcc ccc gtg ccc atg 576 Ser Pro Ser Ser Ala Lys Ser Arg Leu Gln Thr Ala Pro Val Pro Met 180 185 190 cca gac ctg aag aat gtc aag t cc aag atc ggc tcc act gag aac ctg 624 Pro Asp Leu Lys Asn Val Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu 195 200 205 aag cac cag ccg gga ggc ggg aag gtg cag ata att aat aag aag ctg 672 Lys His Gln Pro Gly Gly Gly Lys Val Gln Ile Ile Asn Lys Lys Leu 210 215 220 gat ctt agc aac gtc cag tcc aag tgt ggc tca aag gat aat atc aaa 720 Asp Leu Ser Asn Val Gln Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys 225 230 235 240 cac gtc ccg gga ggc ggc agt gtg caa ata gtc tac aaa cca gtt gac 768 His Val Pro Gly Gly Gly Ser Val Gln Ile Val Tyr Lys Pro Val Asp 245 250 255 ctg agc aag gtg acc tcc aag tgt ggc tca tta aac atc cat cat 816 Leu Ser Lys Val Thr Ser Lys Cys Gly Ser Leu Gly Asn Ile His His 260 265 270 aaa cca gga ggt ggc cag gtg gaa gta aaa tct gag aag ctt gac ttc 864 Lys Pro Gly Gly Gly Gln Val Glu Val Lys Ser Glu Lys Leu Asp Phe 275 280 285 aag gac aga gtc cag tcg aag att ggg tcc ctg gac aat atc acc cac 912 Lys Asp Arg Val Gln Ser Lys Ile Gly Ser Leu Asp Asn Ile Thr His 290 295 300 gtc cct ggc g g ga aat aaa aag att gaa acc cac aag ctg acc ttc 960 Val Pro Gly Gly Gly Asn Lys Lys Ile Glu Thr His Lys Leu Thr Phe 305 310 315 320 cgc gag aac gcc aaa gcc aag aca gac cac ggg gcg gag atc gtg tac 1008 Arg Glu Asn Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile Val Tyr 325 330 335 aag tcg cca gtg gtg tct ggg gac acg tct cca cgg cat ctc agc aat 1056 Lys Ser Pro Val Val Ser Gly Asp Thr Ser Pro Arg His Leu Ser Asn 340 345 350 gtc tcc tcc acc ggc agc atc gac atg gta gac tcg ccc cag ctc gcc 1104 Val Ser Ser Thr Gly Ser Ile Asp Met Val Asp Ser Pro Gln Leu Ala 355 360 365 acg cta gct gac gag gtg tct gcc tcc ctg gcc aag cag ggt ttg tga 1152 Thr Leu Ala Asp Glu Val Ser Ala Ser Leu Ala Lys Gln Gly Leu 370 375 380 <210> 2 <211> 383 <212> PRN <213> Homo sapiens <400> 2 Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly 1 5 10 15 Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His 20 25 30 Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu Lys Ala Glu Glu Ala 35 40 45 Gly Ile Gly Asp Th r Pro Ser Leu Glu Asp Glu Ala Ala Gly His Val 50 55 60 Thr Gln Ala Arg Met Val Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp 65 70 75 80 Asp Lys Lys Ala Lys Gly Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro 85 90 95 Arg Gly Ala Ala Pro Pro Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg 100 105 110 Ile Pro Ala Lys Thr Pro Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly 115 120 125 Glu Pro Pro Lys Ser Gly Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser 130 135 140 Pro Gly Thr Pro Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro 145 150 155 160 Pro Thr Arg Glu Pro Lys Lys Val Ala Val Val Arg Thr Pro Pro Lys 165 170 175 Ser Pro Ser Ser Ala Lys Ser Arg Leu Gln Thr Ala Pro Val Pro Met 180 185 190 Pro Asp Leu Lys Asn Val Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu 195 200 205 Lys His Gln Pro Gly Gly Gly Lys Val Gln Ile Ile Asn Lys Lys Leu 210 215 220 Asp Leu Ser Asn Val Gln Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys 225 230 235 240 His Val Pro Gly Gly Gly Ser Val Gln Ile Val Tyr Lys Pro Val Asp 245 250 255 Leu Ser Lys Val Thr Ser Lys Cys Gly Ser Leu Gly Asn Ile His His 260 265 270 Lys Pro Gly Gly Gly Gln Val Glu Val Lys Ser Glu Lys Leu Asp Phe 275 280 285 Lys Asp Arg Val Gln Ser Lys Ile Gly Ser Leu Asp Asn Ile Thr His 290 295 300 Val Pro Gly Gly Gly Asn Lys Lys Ile Glu Thr His Lys Leu Thr Phe 305 310 315 320 Arg Glu Asn Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile Val Tyr 325 330 335 Lys Ser Pro Val Val Ser Gly Asp Thr Ser Pro Arg His Leu Ser Asn 340 345 350 Val Ser Ser Thr Gly Ser Ile Asp Met Val Asp Ser Pro Gln Leu Ala 355 360 365 Thr Leu Ala Asp Glu Val Ser Ala Ser Leu Ala Lys Gln Gly Leu 370 375 380 <210> 3 <211> 1152 <212> DNA <213> Homo sapiens <400> 3 atg gct gag ccc cgc cag gag ttc gaa gtg atg gaa gat cac gct ggg 48 Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly 1 5 10 15 acg tac ggg ttg ggg gac agg aaa gat cag ggg ggc tac acc atg cac 96 Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His 20 25 30 caa gac caa gag ggt gac acg gac gct ggc ctg aaa gct gaa gaa gca 144 Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu Lys Ala Glu Glu Ala 35 40 45 ggc att gga gac acc ccc agc ctg gaa gac gaa gct gct ggt cac gtg 192 Gly Ile Gly Asp Thr Pro Ser Leu Glu Asp Glu Ala Ala Gly His Val 50 55 60 acc caa gct cgc atg gtc agt aaa agc aaa gac ggg act gga agc gat 240 Thr Gln Ala Arg Met Val Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp 65 70 75 80 gac aaa aaa gcc aag ggg gct gat ggt aaa acg aag atc gcc aca ccg 288 Asp Lys Lys Ala Lys Gly Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro 85 90 95 cgg gga gca gcc cct cca ggc cag aag ggc cag gcc aac gcc acc agg 336 Arg Gly Ala Ala Pro Pro Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg 100 105 110 att cca gca aaa acc ccg ccc gct cca aag aca cca ccc agc tct ggt 384 Ile Pro Ala Lys Thr Pro Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly 115 120 125 gaa cct cca aaa tca ggg gat cgc agc ggc tac agc agc ccc ggc tcc 432 Glu Pro Pro Lys Ser Gly Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser 130 135 140 cca ggc act ccc ggc agc cgc tcc cgc acc ccg tcc ctt cca acc cca Gly Thr Pro Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro 145 150 155 160 ccc acc cgg gag ccc aag aag gtg gca gtg gtc cgt act cca ccc aag 528 Pro Thr Arg Glu Pro Lys Lys Val Ala Val Val Arg Thr Pro Pro Lys 165 170 175 tcg ccg tct tcc gcc aag agc cgc ctg cag aca gcc ccc gtg ccc atg 576 Ser Pro Ser Ser Ala Lys Ser Arg Leu Gln Thr Ala Pro Val Pro Met 180 185 190 cca gac ctg aag aat gtc aag tcc aag atc ggc tcc act gag aac ctg 624 Pro Asp Leu Lys Asn Val Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu 195 200 205 aag cac cag ccg gga ggc ggg aag gtg cag ata att aag aag aag ctg 672 Lys His Gln Pro Gly Gly Gly Lys Val Gln Ile Ile Lys Lys Lys Leu 210 215 220 gat ctt agc aac gtc cag tcc aag tgt ggc tca aag gat aat atc aaa 720 Asp Leu Ser Asn Val Gln Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys 225 230 235 240 cac gtc cc gga ggc ggc agt gtg caa ata gtc tac aaa cca gtt gac 768 His Val Pro Gly Gly Gly Ser Val Gln Ile Val Tyr Lys Pro Val Asp 245 250 255 ctg agc aag gtg acc tcc aag tgt ggc tca tta ggc aac atc cat cat816816 Leu Ser Lys Val Thr Ser Lys Cys Gly Ser Leu Gly Asn Ile His His 260 265 270 aaa cca gga ggt ggc cag gtg gaa gta aaa tct gag aag ctt gac ttc 864 Lys Pro Gly Gly Gly Gln Val Glu Val Lys Ser Glu Lys Leu Asp Phe 275 280 285 aag gac aga gtc cag tcg aag att ggg tcc ctg gac aat atc acc cac 912 Lys Asp Arg Val Gln Ser Lys Ile Gly Ser Leu Asp Asn Ile Thr His 290 295 300 gtc cct ggc gga gga aat aaa aat gaa acc cac aag ctg acc ttc 960 Val Pro Gly Gly Gly Asn Lys Lys Ile Glu Thr His Lys Leu Thr Phe 305 310 315 320 cgc gag aac gcc aaa gcc aag aca gac cac ggg gcg gag atc gtg tac 1008 Arg Glu Asn Ala Lysn Ala Ala Lys Thr Asp His Gly Ala Glu Ile Val Tyr 325 330 335 aag tcg cca gtg gtg tct ggg gac acg tct cca cgg cat ctc agc aat 1056 Lys Ser Pro Val Val Ser Gly Asp Thr Ser Pro Arg His Leu Ser Asn 340 345 350 gtc tcc tcc acc ggc agc atc gac atg gta gac tcg ccc cag ctc gcc 1104 Val Ser Ser Thr Gly Ser Ile Asp Met Val Asp Ser Pro Gln Leu Ala 355 360 365 acg cta gct gac gag gtg tct gcc tcc ctg gcc aag cag gt ttg tga 1152 Thr Leu Ala Asp Glu Val Ser Ala Ser Leu Ala Lys Gln Gly Leu 370 375 380 <210> 4 <211> 383 <212> PRN <213> Homo sapiens <400> 4 Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly 1 5 10 15 Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His 20 25 30 Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu Lys Ala Glu Glu Ala 35 40 45 Gly Ile Gly Asp Thr Pro Ser Leu Glu Asp Glu Ala Ala Gly His Val 50 55 60 Thr Gln Ala Arg Met Val Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp 65 70 75 80 Asp Lys Lys Ala Lys Gly Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro 85 90 95 Arg Gly Ala Ala Pro Pro Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg 100 105 110 Ile Pro Ala Lys Thr Pro Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly 115 120 125 Glu Pro Pro Lys Ser Gly Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser 130 135 140 Pro Gly Thr Pro Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro 145 150 155 160 Pro Thr Arg Glu Pro Lys Lys Val Ala Val Val Arg Thr Pro Pro Lys 165 170 175 Ser Pro Ser Ser Ala Lys Ser Arg Leu Gln Thr Ala Pro Val Pro Met 180 185 190 Pro Asp Leu Lys Asn Val Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu 195 200 205 Lys His Gln Pro Gly Gly Gly Lys Val Gln Ile Ile Lys Lys Lys Leu 210 215 220 Asp Leu Ser Asn Val Gln Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys 225 230 235 240 His Val Pro Gly Gly Gly Ser Val Gln Ile Val Tyr Lys Pro Val Asp 245 250 255 Leu Ser Lys Val Thr Ser Lys Cys Gly Ser Leu Gly Asn Ile His His 260 265 270 Lys Pro Gly Gly Gly Gln Val Glu Val Lys Ser Glu Lys Leu Asp Phe 275 280 285 Lys Asp Arg Val Gln Ser Lys Ile Gly Ser Leu Asp Asn Ile Thr His 290 295 300 Val Pro Gly Gly Gly Asn Lys Lys Ile Glu Thr His Lys Leu Thr Phe 305 310 315 320 Arg Glu Asn Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile Val Tyr 325 330 335 Lys Ser Pro Val Val Ser Gly Asp Thr Ser Pro Arg His Leu Ser Asn 340 345 350 Val Ser Ser Thr Gly Ser Ile Asp Met Val Asp Ser Pro Gln Leu Ala 355 360 365 Thr Leu Ala Asp Glu Val Ser Ala Ser Leu Ala Lys Gln Gly Leu 370 375 380 <210> 5 <211> 1326 <212 > DNA <213> Homo sapiens <400> 5 atg gct gag ccc cgc cag gag ttc gaa gtg atg gaa gat cac gct ggg 48 Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly 1 5 10 15 acg tac ggg ttg ggg gac agg aaa gat cag ggg ggc tac acc atg cac 96 Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His 20 25 30 caa gac caa gag ggt gac acg gac gct ggc ctg aaa gaa tct ccc ctg 144 Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu Lys Glu Ser Pro Leu 35 40 45 cag acc ccc act gag gac gga tct gag gaa ccg ggc tct gaa acc tct 192 Gln Thr Pro Thr Glu Asp Gly Ser Glu Glu Pro Gly Ser Glu Thr Ser 50 55 60 gat gct aag agc act cca aca gcg gaa gat gtg aca gca ccc tta gtg 240 Asp Ala Lys Ser Thr Pro Thr Ala Glu Asp Val Thr Ala Pro Leu Val 65 70 75 80 gat gag gga gct ccc ggc aag cag gct gcc gcg gag ccc cac acg gag 288 Asp Glu Gly Ala Pro Gly Lys Gln Ala Ala Ala Glu Pro His Thr Glu 85 90 95 atc cca gaa gga acc aca gct gaa gaa gca ggc att gga gac acc ccc 336 Ile Pro Glu Gly Thr Thr Ala Glu Glu Ala Gly Ile Gly Asp Thr Pro 100 105 110 agc ctg gaa gac gaa gct gct ggt cac gtg acc caa gct cgc atg gtc 384 Ser Leu Glu Asp Glu Ala Ala Gly His Val Thr Gln Ala Arg Met Val 115 120 125 agt aaa agc aaa gac ggg act gga agc gat gac aaa aaa gcc aag ggg 432 Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp Asp Lys Lys Ala Lys Gly 130 135 140 gct gat ggt aaa acg aag atc gcc aca ccg cgg gga gca gcc cct cca 480 Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro Arg Gly Ala Ala Pro Pro 145 150 155 160 ggc cag aag ggc cag gcc aac gcc acc agg att cca gca aaa acc ccg 528 Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg Ile Pro Ala Lys Thr Pro 165 170 175 ccc gct cca aag aca cca ccc agc tct ggt gaa cct cca aaa tca ggg 576 Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly Glu Pro Pro Lys Ser Gly 180 185 190 gat cgc agc ggc tac agc agc ccc ggc tcc cca ggc act ccc ggc agc 624 Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser 195 200 205 cgc tcc cgc acc ccg tcc ctt cca acc cca ccc acc cgg gag ccc aag 672 Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys 210 215 220 aag gtg gca gtg gtc cgt act cca ccc aag tcg ccg tct tcc gcc aag 720 Lys Val Ala Val Val Arg Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys 225 230 235 240 agc cgc ctg cag aca gcc ccc gtg ccc atg cca gac ctg aag aat gtc 768 Ser Arg Leu Gln Thr Ala Pro Val Pro Met Pro Asp Leu Lys Asn Val 245 250 255 aag tcc aag atc ggc tcc act gag aac ctg aag cac cag ccg gga ggc 816 Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu Lys His Gln Pro Gly Gly 260 265 270 ggg aag gtg cag ata att aat aag aag ctg gat ctt agc aac gtc cag 864 Gly Lys Val Gln Ile Ile Asn Lys Lys Leu Asp Leu Ser Asn Val Gln 275 280 285 tcc aag tgt ggc tca aag gat aat atc aaa cac gtc ccg gga ggc ggc 912 Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys His Val Pro Gly Gly Gly 290 295 300 agt gtg caa ata gtc tac aaa cca gtt gac aagg ag acc tcc 960 Ser Val Gln Ile Val Tyr Lys Pro Val Asp Leu Ser Lys Val Thr Ser 305 310 315 320 aag tgt ggc tca tta ggc aac atc cat cat aaa cca gga ggt ggc cag 1008 Lys Cys Gly Ser Leu Gly Asn Ile His Hi s Lys Pro Gly Gly Gly Gln 325 330 335 gtg gaa gta aaa tct gag aag ctt gac ttc aag gac aga gtc cag tcg 1056 Val Glu Val Lys Ser Glu Lys Leu Asp Phe Lys Asp Arg Val Gln Ser 340 345 350 aag att ggg tcc ctg gac aat atc acc cac gtc cct ggc gga gga aat 1104 Lys Ile Gly Ser Leu Asp Asn Ile Thr His Val Pro Gly Gly Gly Asn 355 360 365 aaa aag att gaa acc cac aag ctg acc ttc cgc gag aac gcc aaa gcc 1152 Lys Ile Glu Thr His Lys Leu Thr Phe Arg Glu Asn Ala Lys Ala 370 375 380 aag aca gac cac ggg gcg gag atc gtg tac aag tcg cca gtg gtg tct 1200 Lys Thr Asp His Gly Ala Glu Ile Val Tyr Lys Ser Pro Val Val Ser 385 390 395 400 ggg gac acg tct cca cgg cat ctc agc aat gtc tcc tcc acc ggc agc 1248 Gly Asp Thr Ser Pro Arg His Leu Ser Asn Val Ser Ser Thr Gly Ser 405 410 415 atc gac atg gta gac tcg ccc cag ctc gcc acg cta gct gac gag gtg 1296 Ile Asp Met Val Asp Ser Pro Gln Leu Ala Thr Leu Ala Asp Glu Val 420 425 430 tct gcc tcc ctg gcc aag cag ggt ttg tga 1326 Ser Ala Ser Leu Ala Lys Gln Gly Leu 435 440 <210> 6 <211> 441 <212> PRN <213> Homo sapiens <400> 6 Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly 1 5 10 15 Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His 20 25 30 Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu Lys Glu Ser Pro Leu 35 40 45 Gln Thr Pro Thr Glu Asp Gly Ser Glu Glu Pro Gly Ser Glu Thr Ser 50 55 60 Asp Ala Lys Ser Thr Pro Thr Ala Glu Asp Val Thr Ala Pro Leu Val 65 70 75 80 Asp Glu Gly Ala Pro Gly Lys Gln Ala Ala Ala Glu Pro His Thr Glu 85 90 95 Ile Pro Glu Gly Thr Thr Ala Glu Glu Ala Gly Ile Gly Asp Thr Pro 100 105 110 Ser Leu Glu Asp Glu Ala Ala Gly His Val Thr Gln Ala Arg Met Val 115 120 125 Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp Asp Lys Lys Ala Lys Gly 130 135 140 Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro Arg Gly Ala Ala Pro Pro 145 150 155 160 Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg Ile Pro Ala Lys Thr Pro 165 170 175 Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly Glu Pro Pro Lys Ser Gly 180 185 190 Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser Pr o Gly Thr Pro Gly Ser 195 200 205 Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys 210 215 220 Lys Val Ala Val Val Arg Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys 225 230 235 240 Ser Arg Leu Gln Thr Ala Pro Val Pro Met Pro Asp Leu Lys Asn Val 245 250 255 Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu Lys His Gln Pro Gly Gly 260 265 270 Gly Lys Val Gln Ile Ile Asn Lys Lys Leu Asp Leu Ser Asn Val Gln 275 280 285 Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys His Val Pro Gly Gly Gly 290 295 300 Ser Val Gln Ile Val Tyr Lys Pro Val Asp Leu Ser Lys Val Thr Ser 305 310 315 320 Lys Cys Gly Ser Leu Gly Asn Ile His His Lys Pro Gly Gly Gly Gln 325 330 335 Val Glu Val Lys Ser Glu Lys Leu Asp Phe Lys Asp Arg Val Gln Ser 340 345 350 Lys Ile Gly Ser Leu Asp Asn Ile Thr His Val Pro Gly Gly Gly Asn 355 360 365 Lys Lys Ile Glu Thr His Lys Leu Thr Phe Arg Glu Asn Ala Lys Ala 370 375 380 Lys Thr Asp His Gly Ala Glu Ile Val Tyr Lys Ser Pro Val Val Ser 385 390 395 400 Gly Asp Thr Ser Pro Arg His Leu Ser Asn Va l Ser Ser Thr Gly Ser 405 410 415 Ile Asp Met Val Asp Ser Pro Gln Leu Ala Thr Leu Ala Asp Glu Val 420 425 430 Ser Ala Ser Leu Ala Lys Gln Gly Leu 435 440 <210> 7 <211> 42 <212> DNA <213> Homo sapiens <400> 7 ggcgggaagg tgcagataat taagaagaag ctggatctta gc 42 <210> 8 <211> 42 <212> DNA <213> Homo sapiens <400> 8 gctaagatcc agcttcttct taattatctg caccttcccg cc 11 <210> 9 <210> 9 <210> 9 40 <212> DNA <213> Artificial Sequence <220><223> A sequence constructed by ligating an XhoI-site sequence to 5'end of the start codon of human tau cDNA. <400> 9 gtggatccct cgagcccatg gctgagcccc gccaggagtt 40 <210 > 10 <211> 28 <212> DNA <213> Homo sapiens <400> 10 gagtcgactt gtcatcgctt ccagtccc 28 <210> 11 <211> 765 <212> DNA <213> Mus musculus <400> 11 atggcgaacc ttggctactg gctgctggcc ctctttgtga gctatgtgg t 60 ctctgcaaaa agcggccaaa gcctggaggg tggaacaccg gtggaagccg gtatcccggg 120 cagggaagcc ctggaggcaa ccgttaccca cctcagggtg gcacctgggg gcagccccac 180 ggtggtggct ggggacaacc ccatggggggacacctct gtagttgg 240 ggtcagcccc atggcggtgg atggggccaa ggagggggta cccataatca gtggaacaag 300 cccagcaaac caaaaaccaa cttcaagcat gtggcagggg ctgcggcagc tggggcagta 360 gtggggggcc ttggtggcta catgctgggg agcgccatga gcaggcccat gatccatttt 420 ggcaacgact gggaggaccg ctactaccgt gaaaacatgt accgctaccc taaccaagtg 480 tactacaggc cagtggatca gtacagcaac cagaacaact tcgtgcacga ctgcgtcaat 540 atcaccatca agcagcacac ggtcgtcacc accaccaagg gggagaactt caccgagacc 600 gatgtgaaga tgatggagcg cgtggtggag cagatgtgcg tcacccagta ccagaaggag 660 tcccaggcct attacgacgg gagaagatcc agcagcaccg tgcttttctc ctcccctcct 720 gtcatcctcc tcatctcctt cctcatcttc ctgatcgtgg gatga 765 <210> 12 <211> 98 <212> DNA <213> Mus musculus <400> 12 gactcctgag tatatttcag aactgaacca tttcaaccga gctgaagcat tctgccttcc 60 tagtggtacc agtccaattt aggagagcca agcagact 98 <210> 13 <211> 29 <212> DNA <213> Mus musculus <400> 13 ggggtacctc gagccttcct gcttgttcc 29 <210> 14 <211> 20 <212> DNA <213> Homo sapiens <400> 14 tccaagatcg gctccactga 20 <210> 15 <211> 28 <212> DNA <21 3> Homo sapiens <400> 15 gtggataccc cctcccccag cctagacc 2
8
【図１】 tau-wild/pET-23dの構造を示す。FIG. 1 shows the structure of tau-wild / pET-23d.
【図２】 tau-wild/pGEX-4Tの構造を示す。FIG. 2 shows the structure of tau-wild / pGEX-4T.
【図３】 tau-T837G/pGEX-4Tの構造を示す。FIG. 3 shows the structure of tau-T837G / pGEX-4T.
【図４】 tau-XhoI-wild/pGEX-4Tの構造を示す。FIG. 4 shows the structure of tau-XhoI-wild / pGEX-4T.
【図５】 tau-XhoI-T837G/pGEX-4Tの構造を示す。FIG. 5 shows the structure of tau-XhoI-T837G / pGEX-4T.
【図６】 phgPrPの作製手順を示すフローチャート。FIG. 6 is a flowchart showing a procedure for producing phgPrP.
【図７】 MoPrP.Xhoの作製手順を示すフローチャー
ト。FIG. 7 is a flowchart showing the procedure for producing MoPrP.Xho.
【図８】 ヒトタウタンパク質の発現を示すウエスタン
ブロット。FIG. 8: Western blot showing expression of human tau protein.
【図９】 タウのリン酸化を示すウエスタンブロット。FIG. 9. Western blot showing phosphorylation of tau.
【図１０】 マウスの運動性を示すグラフ。FIG. 10 is a graph showing mouse motility.
【図１１】 マウスの驚愕反射の強度を示すグラフ。FIG. 11 is a graph showing the intensity of startle reflex in mice.
【図１２】 マウスの驚愕反射のプレパルス阻害を示す
グラフ。FIG. 12 is a graph showing prepulse inhibition of startle reflex in mice.
【図１３】 マウスの受動的回避学習における潜時を示
すグラフ。FIG. 13 is a graph showing latency in passive avoidance learning of mice.
【図１４】 変異型ヒトタウトランスジェニックマウス
の受動的回避学習における潜時短縮を示すグラフ。FIG. 14 is a graph showing latency reduction in passive avoidance learning of mutant human tau transgenic mice.
【図１５】 マウスの水迷路学習における潜時を示すグ
ラフ。FIG. 15 is a graph showing latency in water maze learning of mice.
【図１６】 マウスの能動的回避学習における回避反応
の推移を示すグラフ。FIG. 16 is a graph showing the transition of avoidance reaction in active avoidance learning of mice.
【図１７】 マウスの歯状回の顆粒細胞の代表的トレー
ス。FIG. 17. Representative trace of mouse dentate gyrus granule cells.
【図１８】 マウスにおける長期増強の推移を示すグラ
フ。FIG. 18 is a graph showing changes in long-term potentiation in mice.
【図１９】 マウスにおける持続性筋強直後のスパイク
の平均±標準誤差を示すグラフ。FIG. 19 is a graph showing the mean ± standard error of spikes immediately after continuous muscle strength in mice.
───────────────────────────────────────────────────── フロントページの続き (72)発明者 谷口 泰造 兵庫県神戸市長田区若松町４丁目２番15号 1701 (72)発明者 北村 佳久 兵庫県芦屋市三条町４−８ (72)発明者 森 啓 大阪府大阪市住吉区苅田10−７−21−400 Ｆターム(参考） 4B024 AA01 AA11 BA01 CA04 CA06 DA02 EA04 GA12 HA01 ─────────────────────────────────────────────────── ─── Continued front page (72) Inventor Taizo Taniguchi 4-2-1 Wakamatsucho, Nagata-ku, Kobe City, Hyogo Prefecture 1701 (72) Inventor Yoshihisa Kitamura 4-8 Sanjo-cho, Ashiya-shi, Hyogo (72) Inventor Kei Mori 10-7-21-400 Kanda, Sumiyoshi-ku, Osaka City, Osaka Prefecture F-term (reference) 4B024 AA01 AA11 BA01 CA04 CA06 DA02 EA04 GA12 HA01
Claims (7)
を欠きエクソン１０に対応するアミノ酸を有するアイソ
フォーム（０Ｎ４Ｒアイソフォーム）のヒトタウタンパ
ク質であって、該ヒトタウタンパク質において、野生型
ヒトタウタンパク質の最長アイソフォーム（２Ｎ４Ｒア
イソフォーム）の第279番目のアミノ酸に相当する位置
のアミノ酸がリシンであることを特徴とする変異型ヒト
タウタンパク質をコードする遺伝子を組み込んでなり、
該変異型ヒトタウタンパク質を少なくとも脳において発
現しているトランスジェニックマウス。1. A human tau protein of an isoform (0N4R isoform) having an amino acid sequence corresponding to exon 2 and an amino acid sequence corresponding to exon 10 (0N4R isoform), wherein wild-type human tau protein is used. The longest isoform of (2N4R isoform) of which the amino acid at the position corresponding to the 279th amino acid is lysine, which comprises a gene encoding a mutant human tau protein,
A transgenic mouse expressing the mutant human tau protein in at least the brain.
おいて配列番号４に示すアミノ酸配列を有するものであ
る、請求項１のトランスジェニックマウス。2. The transgenic mouse according to claim 1, wherein the mutant human tau protein has the amino acid sequence represented by SEQ ID NO: 4 in the sequence listing.
示すアミノ酸配列を有するタンパク質をコードするヌク
レオチド配列を有するものである、請求項１又は２のト
ランスジェニックマウス。3. The transgenic mouse according to claim 1 or 2, wherein the gene has a nucleotide sequence encoding a protein having the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing.
ＤＮＡにおいて、野生型ヒトタウタンパク質の最長アイ
ソフォーム（２Ｎ４Ｒアイソフォーム）をコードするＤ
ＮＡの第837番目のヌクレオチドに相当する位置のヌク
レオチドがグアニンであることを特徴とする、請求項１
ないし３の何れかのトランスジェニックマウス。4. A DNA encoding the mutant human tau protein, which encodes the longest isoform of wild-type human tau protein (2N4R isoform)
The nucleotide at the position corresponding to the 837th nucleotide of NA is guanine.
The transgenic mouse of any one of 1 to 3.
ＤＮＡが、配列表において配列番号３に示すヌクレオチ
ド配列を有するものである、請求項１のトランスジェニ
ックマウス。5. The transgenic mouse according to claim 1, wherein the DNA encoding the mutant human tau protein has the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing.
能の低下を示す、請求項１ないし５の何れかのトランス
ジェニックマウス。6. The transgenic mouse according to claim 1, which exhibits a reduced learning ability as compared with a non-transgenic mouse.
遺伝子をヘテロ又はホモで有するものである、請求項１
ないし６のトランスジェニックマウス。7. The heterologous or homozygous gene having the gene encoding the mutant human tau protein.
To 6 transgenic mice.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011043428A (en) * | 2009-08-21 | 2011-03-03 | Taizo Taniguchi | Method for inspecting parkinsonian syndrome using non-human model animal |
| JP2015107972A (en) * | 2007-04-05 | 2015-06-11 | ザ ジェイ．ディヴィッド グラッドストン インスティテューツ | Agents that reduce neuronal overexcitation |
-
2002
- 2002-06-18 JP JP2002176601A patent/JP2003102332A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2015107972A (en) * | 2007-04-05 | 2015-06-11 | ザ ジェイ．ディヴィッド グラッドストン インスティテューツ | Agents that reduce neuronal overexcitation |
| JP2011043428A (en) * | 2009-08-21 | 2011-03-03 | Taizo Taniguchi | Method for inspecting parkinsonian syndrome using non-human model animal |
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