JP6208673B2 - 尋常性乾癬モデルマウス及びその製造方法 - Google Patents
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/12—Animals modified by administration of exogenous cells
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/15—Humanized animals
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/035—Animal model for multifactorial diseases
- A01K2267/0387—Animal model for diseases of the immune system
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Cell Biology (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Description
[1]染色体上にヒトIL-6遺伝子座を保有する、IL-2受容体ガンマ鎖の機能を欠損したNOD/SCIDマウス。
[2]IL-2受容体ガンマ鎖の機能を欠損したNOD/SCIDマウスが、NOD/SCID/IL-2rgKOマウスである、[1]記載のマウス。
[3][1]又は[2]記載のマウスへ、ヒト造血幹細胞を移入すること、及び該マウスを飼育することを含む、免疫系ヒト化マウスの製造方法。
[4]免疫系ヒト化マウスが少なくとも1つの尋常性乾癬の症状を有する、[3]記載の製造方法。
[5]免疫系ヒト化マウスが少なくとも6以上の尋常性乾癬の病理組織像の特徴を有する、[3]記載の製造方法。
[6][3]乃至[5]記載の製造方法により得られる、免疫系ヒト化マウス。
[7]以下の工程を含む、尋常性乾癬の治療又は予防薬の候補化合物のスクリーニング方法:
(1)[6]記載の免疫系ヒト化マウスへ被検物質を投与すること、
(2)被検物質を投与したマウスにおける尋常性乾癬の症状を測定し、該症状を被検物質を投与していない対照マウスにおける尋常性乾癬の症状と比較する工程;及び
(3)上記(2)の比較結果に基づいて、尋常性乾癬の症状を抑制した被検物質を、尋常性乾癬の治療又は予防薬の候補化合物として選択する工程。
本発明は、染色体上にヒトIL-6遺伝子座を保有する、IL-2rg(IL-2受容体ガンマ鎖/コモンガンマ)の機能を欠損したNOD/SCIDマウス(本発明のトランスジェニックマウス)を提供するものである。
本発明は、上記本発明のトランスジェニックマウスへ、ヒト造血幹細胞を移入すること、及び該マウスを飼育することを含む、免疫系ヒト化マウスの製造方法(本発明の製造方法)を提供するものである。
本発明の免疫系ヒト化マウスにおいてはドナーであるヒト由来の免疫系が確立されている。従って、ヒト免疫系のインビボモデルとして、極めて有用である。例えば、本発明の免疫系ヒト化マウスを特定の抗原で免疫することにより、当該抗原に対するヒトの免疫応答を惹起することができ、当該抗原に対するヒト抗体産生や当該抗原を認識するヒトT細胞を誘導することができる。
(1)本発明の免疫系ヒト化マウスへ被検物質を投与すること、
(2)被検物質を投与したマウスにおける尋常性乾癬の症状を測定し、該症状を被検物質を投与していない対照マウスにおける尋常性乾癬の症状と比較する工程;及び
(3)上記(2)の比較結果に基づいて、尋常性乾癬の症状を抑制した被検物質を、尋常性乾癬の治療又は予防薬の候補化合物として選択する工程。
hIL-6TGをコードするヒトBACクローンDNA(BAC clone名, CTD-2594N23, NCBI clone repositoryより入手;GRCh37/hg19の7番染色体22724723-22964038の領域を含み、IL6遺伝子転写領域(RefSeq ID, NM_000600,7番染色体22766766-22771621)を完全にカバーする)を、C57BL/6マウスの受精卵へ導入し、この受精卵から発生したマウスから、ヒトIL-6遺伝子座が染色体上に組み込まれた個体を選択することにより、ヒトIL-6トランスジェニックマウスを樹立した。得られたヒトIL-6トランスジェニックマウスを、NOD/SCID/IL2rgKOマウスへ8回以上交配させることにより、NOD/SCID/IL2rgKO hIL-6トランスジェニックマウスを樹立した。得られたトランスジェニックマウスの各組織におけるヒトIL-6の発現については、qPCRによってRNAレベルでの同定を行った(図1)。RNAレベルの測定のために、それぞれのマウスにリポポリサッカライドを100μg投与し、6時間後に臓器を採材した。採材後、RNAからcDNAを常法により調製し、TaqMan法によりヒトIL-6 mRNA量の定量解析を行った(アプライドバイオシステムズ、ライフテクノロジーズ社)。その結果、リポポリサッカライド処理後のマウス内在性IL-6と同等のレベルのhIL-6 mRNA量の発現が確認できた。同時に、ドナー細胞として、ヒト造血幹細胞が濃縮されたLin-CD34+CD38- 細胞を臍帯血からソーティングによって純化した。ヒトLin-CD34+CD38-細胞を5000-20000個、新生仔NOD/SCID/IL2rgKO hIL-6TGマウスに経静脈的に移植した。新生仔レシピエントマウスが成熟する過程において、フローサイトメトリーによって、CD3、CD19、及びCD33に対するモノクローナル抗体を用いて、マウス骨髄・脾臓において、ヒトT細胞、B細胞、ミエロイド系細胞等が分化していることを確認した(図15〜21)。6ヶ月以上の長期の観察において、HE staining に示されているとおり、皮膚の発赤、硬化を認めた(図3〜9、12)。これらの皮膚病変は、経時的に増悪を認めた(図10)。この病変の病理組織を解析すると、好中球の遺残物、不全角化(parakeratosis)など、乾癬(特に尋常性乾癬)に特徴的病変とヒトT細胞の浸潤を認めた(図6、7、8、9)。このマウスの血清中でのヒト炎症性サイトカインをヒトサイトカインマルチプレックスキット(バイオラッド社、Bio-Plex Pro ヒトサイトカイン27-Plex and 21-Plexアッセイキット)を用いてにて測定したところ、各種ヒト炎症性サイトカインの上昇を認めた(図13)。図13において、サンプル1、2、3は、それぞれhIL-6 TGマウス、非遺伝子導入ヒト化マウス1、2を示している。更に、hIL-6 TGマウスへの皮膚炎発症時の各種臓器におけるヒトサイトカイン、ケモカイン遺伝子のmRNA量の変化を定量PCR法(TaqMan法、アプライドバイオサイエンス、ライフテクノロジーズ社)で計測した。384-Well Micro Fluidic Cards(アプライドバイオシステムズ、ライフテクノロジーズ社)を用いて、TaqMan法により96種類のヒト遺伝子発現量を調べた。測定したヒト遺伝子名とそれぞれに用いたTaqManプローブのリストを表1に示す。図14は、非遺伝子導入ヒト化マウス(対照群)に対するIL6トランスジェニックヒト化マウスの皮膚炎発症時の遺伝子発現変化をノーマライズされた検出限界サイクル数の変化(-ΔΔCt値)としてヒートマップにより示したものである(7臓器:GS、顎下腺;Brain、脳;Liver、肝臓;Skin(back)、皮膚(背中);Lung、肺;Spleen、脾臓;AXLN、 腋窩リンパ節)。ほとんどの臓器でヒト炎症性サイトカイン・ケモカインmRNA量の顕著な増加が見られた。
実施例1と同様に、NOD/SCID/IL2rgKO hIL-6トランスジェニックマウスへ、臍帯血から単離したヒトLin-CD34+CD38-細胞を移植し、移植したレシピエントマウスにおける、尋常性乾癬の症状(脱毛)の発症時期をモニターした。結果を表2に示す。
Claims (6)
- 染色体上にヒトIL-6遺伝子座を保有する、IL-2受容体ガンマ鎖の機能を欠損したNOD/SCIDマウス。
- IL-2受容体ガンマ鎖の機能を欠損したNOD/SCIDマウスが、NOD/SCID/IL-2rgKOマウスである、請求項1記載のマウス。
- 請求項1又は2記載のマウスへ、ヒト造血幹細胞を移入すること、及び該マウスを飼育することを含む、免疫系ヒト化マウスの製造方法。
- 免疫系ヒト化マウスが少なくとも1つの尋常性乾癬の症状を有する、請求項3記載の製造方法。
- 免疫系ヒト化マウスが少なくとも6以上の尋常性乾癬の病理組織像の特徴を有する、請求項3記載の製造方法。
- 以下の工程を含む、尋常性乾癬の治療又は予防薬の候補化合物のスクリーニング方法:
(1)請求項3乃至5のいずれか1項記載の製造方法により得られる免疫系ヒト化マウスへ被検物質を投与すること、
(2)被検物質を投与したマウスにおける尋常性乾癬の症状を測定し、該症状を被検物質を投与していない対照マウスにおける尋常性乾癬の症状と比較する工程;及び
(3)上記(2)の比較結果に基づいて、尋常性乾癬の症状を抑制した被検物質を、尋常性乾癬の治療又は予防薬の候補化合物として選択する工程。
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