JP2008099601A - Transgenic non-human animal - Google Patents
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Abstract
Description
本発明は、オートファジーの実行部隊の一員と認識されているＬＣ３ホモログであるＧＡＢＡＲＡＰ又はＧＡＴＥ１６の修飾体を発現しているトランスジェニック非ヒト動物又はその子孫に関する。 The present invention relates to a transgenic non-human animal or a progeny thereof expressing a modified form of GABARAP or GATE16, which is an LC3 homolog recognized as a member of an execution unit of autophagy.
オートファジーは、隔離膜が伸長しオルガネラを含む細胞質成分を取り囲んだ脂質二重膜構造体がリソソームと融合し、その内容物をリソソーム内の消化酵素が分解する細胞内大規模分解経路である。このオートファジーの因子が全身で欠損したマウスは新生児致死性である（非特許文献１）。脳において欠損すると、神経変性疾患になり（非特許文献２）、肝臓において欠損すると、肝肥大を伴う肝炎になる（非特許文献２）。また、ガン細胞においては、オートファジーに関する遺伝子のいくつかが減少しているとの報告がある（非特許文献３）。 Autophagy is a large-scale intracellular degradation pathway in which a lipid bilayer structure that extends a sequestering membrane and surrounds cytoplasmic components including organelles fuses with lysosomes and digests the digestion enzymes in the lysosomes. Mice deficient in autophagy factors throughout the body are neonatal lethal (Non-patent Document 1). When deficient in the brain, it becomes a neurodegenerative disease (Non-patent Document 2), and when deficient in the liver, it results in hepatitis with liver hypertrophy (Non-patent Document 2). In cancer cells, there are reports that some of genes related to autophagy are decreased (Non-patent Document 3).
ＬＣ３（microtubule associated protein 1A light chain3）は微小管関連タンパク質の１つとして、脳から単離されてきた（非特許文献４、５）。その後の研究でＬＣ３は、オートファジーの際のモディファイヤーとしてＡｐｇ７とＡｐｇ３によってスビキチン様修飾をうけて細胞質型ＬＣ３（ＬＣ３−Ｉ）から膜結合型ＬＣ３（ＬＣ３−II）になり、オートファゴソーム膜上に局在することがわかってきた（非特許文献６〜９）。かかる点から、ＬＣ３は、オートファジーの実行部隊の一員と認識されるに至っている。 LC3 (microtubule associated protein 1A light chain 3) has been isolated from the brain as one of microtubule-associated proteins (Non-Patent Documents 4 and 5). In subsequent studies, LC3 was converted from cytoplasmic LC3 (LC3-I) to membrane-bound LC3 (LC3-II) by undergoing subiquitin-like modification with Apg7 and Apg3 as a modifier during autophagy, and on the autophagosome membrane. (Non-patent documents 6 to 9). From this point, LC3 has come to be recognized as a member of the execution unit of autophagy.
一方、ＧＡＢＡＲＡＰは神経伝達に関わるＧＡＢＡ受容体と結合する因子として脳において発見されたが、これがオートファジー因子であるＬＣ３のホモログであった。in vitroではＬＣ３と同様の修飾を受ける事がわかっているが、細胞内動態の解析は全くされていない。脳においてオートファジーを欠損させたマウスでは、神経変性疾患様表現型を示す（非特許文献２）。この時、このマウスではＧＡＢＡＲＡＰの発現も上昇していた。従って、脳における神経変性疾患に関してはＧＡＢＡＲＡＰが関与している可能性がある。 On the other hand, GABARAP was found in the brain as a factor that binds to a GABA receptor involved in neurotransmission, and this was a homolog of LC3, which is an autophagy factor. In vitro, it is known that it undergoes the same modification as LC3, but intracellular dynamics have not been analyzed at all. Mice deficient in autophagy in the brain show a neurodegenerative disease-like phenotype (Non-Patent Document 2). At this time, GABARAP expression was also increased in this mouse. Therefore, GABARAP may be involved in neurodegenerative diseases in the brain.
また、ＧＡＴＥ１６は、神経伝達物質などを細胞外に輸送する経路の輸送を促進する因子として脳より単離された。これがオートファジー因子であるＬＣ３のホモログであった。in vitroではＬＣ３と同様の修飾を受ける事がわかっているが、細胞内動態の解析は全くされていない。また、肝臓においてオートファジーを欠損させると、ＧＡＴＥ１６の発現量も増加する。従って、ＧＡＴＥ１６がオートファジーの欠損により引き起こされる、病態に関与している可能性がある。
このようにオートファジーは、種々の生理機能や疾患に関与していることが判明しており、注目されているが、これまでオートファジーに着目した医薬の開発はなされていない。
従って、本発明の目的は、オートファジーの研究及びオートファジーに関与する疾患の治療薬のスクリーニングに用いることのできるモデル動物を提供することにある。
Thus, autophagy has been found to be involved in various physiological functions and diseases, and has attracted attention. However, no medicine has been developed so far that focuses on autophagy.
Accordingly, an object of the present invention is to provide a model animal that can be used for research of autophagy and screening for therapeutic agents for diseases involved in autophagy.
そこで本発明は、ＧＡＢＡＲＡＰ及びＧＡＴＥ１６に着目して研究してきたところ、これらのアミノ末端にＧＦＰを結合した修飾ＧＡＢＡＲＡＰ又は修飾ＧＡＴＥ１６を発現する遺伝子を導入したトランスジェニック非ヒト動物の作製に成功し、このトランスジェニック動物が全身に修飾ＧＡＢＡＲＡＰ又は修飾ＧＡＴＥ１６を発現しており、オートファジーが関与する疾患の研究モデルとして、又は医薬のスクリーニングモデルとして有用であることを見出し、本発明を完成した。 Therefore, the present invention has been studied focusing on GABARAP and GATE16. As a result, the present inventors have succeeded in producing a transgenic non-human animal into which a gene expressing a modified GABARAP or a modified GATE16 in which GFP is bound to the amino terminus thereof. The present inventors have found that a transgenic animal expresses modified GABARAP or modified GATE16 throughout the body and is useful as a research model for diseases involving autophagy or as a pharmaceutical screening model, thereby completing the present invention.
すなわち、本発明は、ＧＡＢＡＲＡＰ又はＧＡＴＥ１６のアミノ末端にＧＦＰが結合した修飾ＧＡＢＡＲＡＰ又は修飾ＧＡＴＥ１６を全身に発現しているトランスジェニック非ヒト動物又はその子孫を提供するものである。
また、本発明は上記トランスジェニック非ヒト動物又はその子孫に被験物質を投与し、ＧＡＢＡＲＡＰ又はＧＡＴＥ１６が関与する疾患の治療薬のスクリーニング方法を提供するものである。
That is, the present invention provides a transgenic non-human animal or its progeny expressing systemically modified GABARAP or modified GATE16 in which GFP is bound to the amino terminus of GABARAP or GATE16.
The present invention also provides a method for screening a therapeutic agent for a disease involving GABARAP or GATE16, by administering a test substance to the transgenic non-human animal or its progeny.
本発明のトランスジェニック非ヒト動物を用いれば、オートファジーが関与する疾患の研究、各種疾患におけるオートファジーの役割の研究、さらにはオートファジーが関与する疾患の治療薬の検索が可能になる。 By using the transgenic non-human animal of the present invention, it becomes possible to study diseases related to autophagy, study the role of autophagy in various diseases, and search for therapeutic agents for diseases related to autophagy.
本発明のトランスジェニック非ヒト動物は、アミノ末端にＧＦＰが結合した修飾ＧＡＢＡＲＡＰ又は修飾ＧＡＴＥ１６を全身に発現しているものである。ここでＧＡＢＡＲＡＰ及びＧＡＴＥ１６遺伝子は、すでにクローニングされている（Tanida et al, J. Biol. Chem. 2001；276(3)：1701-1706, Tanida et al, Biochem. Biophys. Res. Commun. 2002, Sep 6；296(5)：1164-1170、Gene Bank Accession No.AF161586）。GFP(Green Fluorescent Protein)は、オワンクラゲがもつ分子量約２７kpaの蛍光タンパク質であり、この遺伝子もすべてクローニングされている（Prasher et al. Gene. 1992 Feb 15;111(2):229-33、Gene Bank Accession No.DQ893101）。 The transgenic non-human animal of the present invention expresses modified GABARAP or modified GATE16 in which GFP is bound to the amino terminus throughout the body. Here, GABARAP and GATE16 genes have already been cloned (Tanida et al, J. Biol. Chem. 2001; 276 (3): 1701-1706, Tanida et al, Biochem. Biophys. Res. Commun. 2002, Sep. 6; 296 (5): 1164-1170, Gene Bank Accession No. AF161586). GFP (Green Fluorescent Protein) is a fluorescent protein with a molecular weight of about 27 kpa possessed by Aequorea jellyfish, and all of this gene has also been cloned (Prasher et al. Gene. 1992 Feb 15; 111 (2): 229-33, Gene Bank Accession No. DQ893101).
本発明のトランスジェニック非ヒト動物は、例えば前記修飾ＧＡＢＡＲＡＰ又は修飾ＧＡＴＥ１６遺伝子を組み込んだＤＮＡ（組み換えＤＮＡ）常法に従い非ヒト動物に導入することにより作製できる。この組み換えＤＮＡは、ＧＦＰをコードする遺伝子をＧＡＢＡＲＡＰｃＤＮＡ又はＧＡＴＥ１６ｃＤＮＡのセンス方向の上流に導入することにより得られる。ここで、組み換えＤＮＡには、非ヒト動物において当該修飾ＧＡＢＡＲＡＰ又は修飾ＧＡＴＥ１６を発現させるための適切なプロモータを用いることができる。このようなプロモータとしては、サイトメガロウィルス（ＣＭＶ）プロモータ、ＳＶ４０プロモータ、ＥＦ１−αプロモータ等が挙げられる。 The transgenic non-human animal of the present invention can be prepared, for example, by introducing the modified GABARAP or modified GATE16 gene into a non-human animal according to a conventional method (DNA recombinant DNA). This recombinant DNA can be obtained by introducing a gene encoding GFP upstream of the sense direction of GABARAP cDNA or GATE16 cDNA. Here, as the recombinant DNA, an appropriate promoter for expressing the modified GABARAP or the modified GATE16 in a non-human animal can be used. Examples of such a promoter include cytomegalovirus (CMV) promoter, SV40 promoter, EF1-α promoter, and the like.
かくして得られる組み換えＤＮＡを導入するための非ヒト哺乳動物としては、ウシ、ブタ、ヒツジ、ヤギ、ウサギ、イヌ、ネコ、モルモット、ハムスター、ラット、マウス等が挙げられる。好ましくは、ウサギ、イヌ、ネコ、モルモット、ハムスター、マウス又はラットであり、なかでもモルモット、ハムスター、マウス、ラット等の齧歯目（Rodentia）が好ましく、とりわけマウスが好ましい。 Examples of the non-human mammal for introducing the recombinant DNA thus obtained include cattle, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice and the like. Preferred are rabbits, dogs, cats, guinea pigs, hamsters, mice or rats, among which rodents such as guinea pigs, hamsters, mice and rats are preferred, and mice are particularly preferred.
本発明のトランスジェニック動物は、例えば非ヒト哺乳動物の受精卵に、前記組み換えＤＮＡを導入し、当該受精卵を当該動物の雌に着床させることにより作製される。ここで、受精卵としては、雄精前核時期（受精後約１２時間位）のものが好ましい。また組み換えＤＮＡの導入方法としては、リン酸カルシウム法、電気パルス法、リポフェクション法、凝集法、マイクロインジェクション法、パーティクルガン法、ＤＥＡＥ−デキストラン法等が挙げられるが、マイクロインジェクション法が特に好ましい。 The transgenic animal of the present invention is produced, for example, by introducing the recombinant DNA into a fertilized egg of a non-human mammal and implanting the fertilized egg into a female of the animal. Here, as a fertilized egg, a male prosperous nucleus time (about 12 hours after fertilization) is preferable. Examples of the method for introducing recombinant DNA include the calcium phosphate method, the electric pulse method, the lipofection method, the aggregation method, the microinjection method, the particle gun method, the DEAE-dextran method, and the microinjection method is particularly preferable.
組み換えＤＮＡを導入した受精卵は、当該受精卵と同種の動物の雌に着床させる。着床の手段は、偽妊娠雌性動物の卵管に人工的に移植、着床させる手段が好ましい。かくして、受精卵を着床させた動物から生まれた仔の中から、目的とする遺伝子を発現している個体を選別し、当該個体を継代すればよい。 The fertilized egg into which the recombinant DNA has been introduced is implanted into female females of the same species as the fertilized egg. The means for implantation is preferably a means for artificial implantation and implantation in the oviduct of a pseudopregnant female animal. Thus, an individual expressing a target gene may be selected from offspring born from animals implanted with fertilized eggs, and the individual may be passaged.
得られたトランスジェニック動物に目的遺伝子が含まれているか否かの確認は、ＤＮＡを採取し、ポリメラーゼ連鎖反応（ＰＣＲ）及びサザンブロッティング法による導入遺伝子の解析によって行うことができる。 Whether or not the target gene is contained in the resulting transgenic animal can be confirmed by collecting DNA and analyzing the transgene by polymerase chain reaction (PCR) and Southern blotting.
また得られたトランスジェニック動物における修飾ＧＡＢＡＲＡＰ又は修飾ＧＡＴＥ１６の発現は、臓器ごとに細胞破砕液を調製し、抗ＧＦＰ抗体を用いてウェスタンブロット法により確認できる。 Moreover, the expression of the modified GABARAP or the modified GATE16 in the obtained transgenic animal can be confirmed by preparing a cell lysate for each organ and using Western blotting using an anti-GFP antibody.
本発明のトランスジェニック動物は、全身に修飾ＧＡＢＡＲＡＰ又は修飾ＧＡＴＥ１６が発現していることから、ＧＡＢＡＲＡＰ又はＧＡＴＥ１６をコントロールする因子をスクリーニングでき、オートファジーが関与する疾患の研究、例えば神経変性疾患、肝臓疾患、ガン化等に関与する因子のスクリーニングに用いることができる。 Since the transgenic animal of the present invention expresses modified GABARAP or modified GATE16 throughout the body, it is possible to screen for factors that control GABARAP or GATE16, and research on diseases involving autophagy, such as neurodegenerative diseases and liver diseases. It can be used for screening for factors involved in canceration.
また、本発明のトランスジェニック動物に被験物質を投与し、ＧＡＢＡＲＡＰ又はＧＡＴＥ１６の増域を評価すれば、ＧＡＢＡＲＡＰ又はＧＡＴＥ１６が関与する疾患の治療薬をスクリーニングすることができる。ＬＣ３が関与する疾患としては、神経変性疾患（例えばアルツハイマー症、パーキンソン病、ハンチントン舞踏病）、肝炎（急性肝炎、慢性肝炎）、肝硬変、ガン、感染症、免疫異常等が挙げられる。 In addition, if a test substance is administered to the transgenic animal of the present invention and the increase of GABARAP or GATE16 is evaluated, a therapeutic drug for a disease involving GABARAP or GATE16 can be screened. Examples of diseases involving LC3 include neurodegenerative diseases (eg, Alzheimer's disease, Parkinson's disease, Huntington's disease), hepatitis (acute hepatitis, chronic hepatitis), cirrhosis, cancer, infectious diseases, immune abnormalities, and the like.
次に実施例を挙げて本発明をさらに詳細に説明するが、本発明は何らこれに限定されるものではない。 EXAMPLES Next, although an Example is given and this invention is demonstrated further in detail, this invention is not limited to this at all.
実施例１
（１）ＧＦＰ−ｔａｇの導入：合成オリゴヌクレオチドとＰＣＲ法を用いて、ＧＡＢＡＲＡＰ ｃＤＮＡのセンス方向の上流（タンパク質のアミノ酸末端の上流）に、ＧＦＰ（green fluorescent protein）をコードする塩基配列を導入した（ＧＦＰ−ＧＡＢＡＲＡＰ ＤＮＡ断片）。
Example 1
(1) Introduction of GFP-tag: A base sequence encoding GFP (green fluorescent protein) was introduced upstream of the sense direction of GABARAP cDNA (upstream of the amino acid terminal of the protein) using a synthetic oligonucleotide and PCR method. (GFP-GABARAP DNA fragment).
（２）得られたＧＦＰ−ＧＡＢＡＲＡＰ ＤＮＡ断片をＣＭＶ（サイトメガロウィルス）プロモーターの下流に導入した。作成した［ＣＭＶプロモーター−ＧＦＰ−ＧＡＢＡＲＡＰ ＤＮＡ］断片を用いて、定法に従い、ＧＦＰ−ＧＡＢＡＲＡＰトランスジェニックマウスを作成した。その後、遺伝的に安定させるために、野生型Ｂ６Ｊマウスと交配を続け、世代間を安定的にＧＦＰ−ＧＡＢＡＲＡＰ発現用ＤＮＡ断片が遺伝していくのをＰＣＲ法を用いたgenotypingにより確認した。具体的には、まずマウス尾部より定法に従い、ゲノムDNAを抽出し、そのDNAを鋳型にCAGSFプライマー（5'-GGCTTCTGGCGTGTGACC-3'：配列番号１）とGABARAPRvプライマー（5'-TCACAGACCGTAGACAC-3'：配列番号２）を用いて、ＫＯＤ−ｐｌｕｓ−ＤＮＡ ｐｏｌｙｍｅｒａｓｅ酵素により、目的の断片約４００ｂｐを増幅していることで、遺伝子型を判定した。 (2) The obtained GFP-GABARAP DNA fragment was introduced downstream of the CMV (cytomegalovirus) promoter. Using the prepared [CMV promoter-GFP-GABARAP DNA] fragment, a GFP-GABARAP transgenic mouse was prepared according to a conventional method. Thereafter, in order to make it genetically stable, mating with wild-type B6J mice was continued, and it was confirmed by genotyping using the PCR method that the DNA fragment for GFP-GABARAP expression was stably inherited between generations. Specifically, genomic DNA is first extracted from the mouse tail according to a conventional method, and the CAGSF primer (5'-GGCTTCTGGCGTGTGACC-3 ': SEQ ID NO: 1) and GABARAPRv primer (5'-TCACAGACCGTAGACAC-3': Using SEQ ID NO: 2), about 400 bp of the target fragment was amplified by the KOD-plus-DNA polymerase enzyme, and the genotype was determined.
（３）臓器におけるＧＦＰ−ＧＡＢＡＲＡＰタンパク質の発現は、定法に従って臓器ごとにダウンス型ホモジナイザーで組織を破壊後、１％ＳＤＳ存在下でタンパク質を抽出し、遠心によ不溶物質を取り除くことで、細胞破砕液を調製し、１２．５％ＳＤＳ−ＰＡＧＥによりタンパク質を分子量ごとに分離後、抗ＧＦＰ抗体を用いてウエスタンブロット法により確認できた。 (3) The expression of the GFP-GABARAP protein in the organ is performed by disrupting the tissue with a Dounce homogenizer for each organ according to a conventional method, extracting the protein in the presence of 1% SDS, and removing the insoluble material by centrifugation, thereby disrupting the cell. The solution was prepared, and the protein was separated by molecular weight by 12.5% SDS-PAGE, and then confirmed by Western blotting using an anti-GFP antibody.
ＧＦＰ−ＧＡｂＡＲＡＰｔｇマウス各臓器におけるＧＦＰ−ＧＡＢＡＲＡＰの発現を調べた。調べたすべての臓器において、目的タンパク質の発現を認められた（図２）。 The expression of GFP-GABARAP in each organ of GFP-GAbARAPtg mice was examined. Expression of the target protein was observed in all organs examined (FIG. 2).
また、マウス組織ををホルムアミド存在下で固定し、超薄切片を調製ご、臓器ごとに蛍光顕微鏡を用いた観察を行い、各臓器に発現している事を確認した（図３）。 In addition, mouse tissues were fixed in the presence of formamide, ultrathin sections were prepared, and each organ was observed using a fluorescence microscope to confirm that it was expressed in each organ (FIG. 3).
実施例２
（１）ＧＦＰ−ｔａｇの導入：合成オリゴヌクレオチドとＰＣＲ法を用いてＧＡＴＥ１６ ｃＤＮＡのセンス方向の上流（タンパク質のアミノ酸末端の上流）に、ＧＦＰ(green fluorescent protein)をコードする塩基配列を導入した（ＧＦＰ−ＧＡＴＥ１６ＤＮＡ断片）。
Example 2
(1) Introduction of GFP-tag: A nucleotide sequence encoding GFP (green fluorescent protein) was introduced upstream of the sense direction of GATE16 cDNA (upstream of the amino acid terminal of the protein) using a synthetic oligonucleotide and PCR method ( GFP-GATE16 DNA fragment).
得られたＧＦＰ−ＧＡＴＥ１６ＤＮＡ断片をＣＭＶ（サイトメガロウィルス）プロモーターの下流に導入した。作成した［ＣＭＶプロモーター−ＧＦＰ−ＧＡＴＥ１６ＤＮＡ］断片を用いて、定法に従い、ＧＦＰ−ＧＡＴＥ１６トランスジェニックマウスを作成した。その後、遺伝的に安定させるために、野生型Ｂ６Ｊマウスと交配を続け、世代間を安定的にＧＦＰ−ＧＡＴＥ１６発現用ＤＮＡ断片が遺伝していくのをＰＣＲ法を用いたgenotypingにより確認した。具体的には、まずマウス尾部より定法に従い、ゲノムＤＮＡを抽出し、そのＤＮＡを鋳型にＣＡＧＳＦプライマー（5'-GGCTTCTGGCGTGTGACC-3'：配列番号１）とG16Rvプライマー（5'- TCAGAAGCCAAAAGTGTTC-3' ：配列番号２）を用いて、ＫＯＤ−ｐｌｕｓ−ＤＮＡ ｐｏｌｙｍｅｒａｓｅ酵素により、目的の断片約４００ｂｐを増幅していることで、遺伝子型を判定した。 The obtained GFP-GATE16 DNA fragment was introduced downstream of the CMV (cytomegalovirus) promoter. Using the prepared [CMV promoter-GFP-GATE16 DNA] fragment, a GFP-GATE16 transgenic mouse was prepared according to a conventional method. Thereafter, in order to genetically stabilize, the mating with wild-type B6J mice was continued, and it was confirmed by genotyping using the PCR method that the DNA fragment for GFP-GATE16 expression was stably inherited between generations. Specifically, first, genomic DNA is extracted from a mouse tail according to a conventional method, and CAGSF primer (5'-GGCTTCTGGCGTGTGACC-3 ': SEQ ID NO: 1) and G16Rv primer (5'-TCAGAAGCCAAAAGTGTTC-3': Using SEQ ID NO: 2), about 400 bp of the target fragment was amplified by the KOD-plus-DNA polymerase enzyme, and the genotype was determined.
（３）臓器におけるＧＦＰ−ＧＡＴＥ１６タンパク質の発現は、定法に従って臓器ごとに細胞破砕液を調製し、抗ＧＦＰ抗体を用いてウエスタンブロット法により確認できた。 (3) Expression of GFP-GATE16 protein in the organ was confirmed by Western blotting using an anti-GFP antibody by preparing a cell disruption solution for each organ according to a conventional method.
また、臓器ごとに蛍光顕微鏡を用いた観察を行い、各臓器に発現している事を確認した（図６）。 Moreover, the observation using a fluorescence microscope was performed for each organ, and it was confirmed that it was expressed in each organ (FIG. 6).
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| CN114107401A (en) * | 2021-11-12 | 2022-03-01 | 中国人民解放军海军军医大学 | Construction method and application of transgenic non-human animal |
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| CN114107401A (en) * | 2021-11-12 | 2022-03-01 | 中国人民解放军海军军医大学 | Construction method and application of transgenic non-human animal |
| CN114107401B (en) * | 2021-11-12 | 2024-03-22 | 中国人民解放军海军军医大学 | Construction method and application of transgenic non-human animal |
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