JP6650649B2 - 遺伝子改変非ヒトモデル動物 - Google Patents
遺伝子改変非ヒトモデル動物 Download PDFInfo
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Description
背景技術
1.チオレドキシンをコードする遺伝子が改変された非ヒトモデル動物。
2.チオレドキシンをコードする改変された遺伝子が、基質タンパク質のジスルフィド結合を還元するために消費される単位時間あたりのNADPH(ニコチンアミドアデニンジヌクレオチドリン酸)量を減少させ、その減少割合が10%から90%の範囲にあることを特徴とする、前項1に記載の非ヒトモデル動物。
3.老化、腎臓病、心血管疾患、高血圧、大動脈解離、慢性閉塞性肺疾患、年齢依存性てんかん、脂質代謝異常、貧血、骨粗鬆症および免疫異常からなる群から選択されるいずれかの疾患にモデルとして適応される、前項1または2に記載の非ヒトモデル動物。
4.前項1〜3のいずれか1に記載の疾患モデルを用いることを特徴とする薬剤のスクリーニング方法。
ATGGTGAAGCTGATCGAGAGCAAGGAAGCTTTTCAGGAGGCCCTGGCCGCTGCGGGAGACAAGCTTGTGGTAGTGGACTTCTCTGCCACGTGGTGTGGACCTTGCAAAATGATCAAGCCCTTCTTTCATTCCCTCTGTGACAAGTATTCCAATGTGGTGTTCCTTGAAGTAGACGTGGATGACTGCCAGGATGTTGCTGCAGACTGTGAAGTCAAATGCATGCCGACCTTCCAGTTCTATAAAAAGGGTCAAAAGGTTGGGGAGTTCTCTGGTGCTAACAAGGAAAAGCTCGAAGCCACTATTACGGAGTTTGCCTAA
(配列番号2)
MVKLIESKEAFQEALAAAGDKLVVVDFSATWCGPCKMIKPFFHSLCDKYSNVVFLEVDVDDCQDVAADCEVKCMPTFQFYKKGQKVGEFSGANKEKLEATITEFA
本実施例では、表現型異常を示すラットの解析を行い、当該解析結果に基づくTxn1遺伝子改変ラットの作製について説明する。
ゲノム上にランダムに点突然変異を誘発する化学変異原エチルニトロソウレア(N-ethyl-N-nitorosourea;ENU)(40mg/kg)を雄ラットF344/NSlcに2回(9, 10週齢時)腹腔内投与し、雌ラットF344/NSlcと交配することで、仔ラット群を作製した。仔ラット群の中から表現型異常を示すラットを選別し、表現異常に関わる遺伝子を検索した。遺伝子の解析として、野生型との交配により戻し交雑群を作製し、全染色体上のDNA多型マーカーを用いて病型との連鎖解析を行った。これにより表現型異常に関わる関連座位を絞り込み、次世代シークエンサーを用いたゲノムシークエンス解析により、原因遺伝子はTxn1遺伝子の変異であることを同定した。図1は、自動キャピラリーシークエンサー(ABI3100)を用いたシークエンス解析により、当該ラットのチオレドキシンTxn1遺伝子変異を解析した結果を示す。当該表現型異常を示すラットはTxn1遺伝子の第54番目のコドンTTCがCTCへ変化しており、その結果第54番目のアミノ酸がFからLに置換していることが確認された(F54L)。
上記1)において確認されたTxn1遺伝子の第54番目のコドンTTCがCTCへ変化したラットについて、10世代以上同系統の野生型(Wild Type:WT)と戻し交配を行い、遺伝的バックグラウンドをWTに一致させ、Txn1遺伝子改変ラット(ヘテロ接合体:Hetero)を作製した。当該ヘテロ接合体のTxn1遺伝子改変ラット同士を交配することにより、ホモ接合体のTxn1遺伝子改変ラットとヘテロ接合体のTxn1遺伝子改変ラットおよび野生型ラット(WT)を得た。以下の実験では、この同胞のWTをコントロールとして使用した。以降において、ホモ接合体の変異ラットを「Homo」といい、ヘテロ接合体の変異ラットを「Hetero」ということとする。また、野生型ラット(WT)は「WT」という。以下の実験例においても同様である。
実施例1で作製したTxn1遺伝子改変ラットについて、Txn1蛋白質の発現を確認した。生後1ヶ月、4ヶ月のWT、Hetero、Homo、および、生後12ヶ月のWT、Heteroから腎臓、心臓、肝臓、脳の組織を摘出後、液体窒素で凍結し、超低温冷凍庫に保管した。各組織よりLysis Bufferにて蛋白質を抽出後、超音波処理を行った。冷却遠心機にて沈殿物を除去し、蛋白質濃度を測定後、抗Txn1抗体、および、インターナルコントロールである抗GAPDH抗体を用いたウエスタンブロットを行なった。WTに比し、Hetero、Homoラットの各組織におけるTxn1蛋白質は発現量に大きな差がないことが明らかになった。このことより、変異型Txn1蛋白質も分解されることなく安定に発現していると考えられた。
本実験例では、実施例1で作製したHomoラット、および、WTラットのTxn1遺伝子cDNAを用いてTxn1組換え蛋白質を作成し、Txn1活性の測定を行なった。WT、および、Homoラットの組織からRNAを抽出後、RT-PCRにて正常型(WT)、および、変異型(F54L)のcDNAを作成し、FLAG-tagを持つpCMV-Tag2Bベクターに挿入した。また、Txn1の活性中心である35番目のシステインをセリンに変異させた変異型C35Sを作成しpCMV-Tag2Bベクターに導入した。作成したプラスミドは全てシークエンス解析により配列を確認した。各ベクター(WT、F54L、C35S、 Empty vector)をヒト正常培養細胞HEK293に導入し遺伝子発現させた後、抗FLAG 抗体 ビーズ(Sigma;ANTI-FLAG M2 Agarose Affinity Gel、A2220)を用いて、FLAG融合型チオレドキシン蛋白を精製した。蛋白濃度を定量し、抗Txn1抗体(Cell Signaling Technology, #2298)、抗FLAG抗体(Sigma, F3165)を用いたウエスタンブロットにて各蛋白量を比較確認した後、 Thioredoxin Activity Assay Kit (Redoxica, TR-7011K)と微量分光光度計(DeNovix社、 DS-11+)を用いてTxn1の活性測定を行なった。
本実験例では、実施例1で作製したHomo、Hetero、および、野生型WTラットの組織より蛋白質を抽出し、Txn1の活性測定を行なった。 生後4ヶ月のHomo、Hetero、WTの組織を摘出後、液体窒素で凍結し、超低温冷凍庫に保管した。Lysis Bufferを用いて腎臓組織から蛋白を抽出し超音波処理を行った後、冷却遠心機にて沈殿物を除去し蛋白濃度を定量した。抗Txn1抗体(Cell Signaling Technology, #2298)を用いたウエスタンブロットにて蛋白量を確認した後、Thioredoxin Activity Assay Kit (Redoxica, TR-7011K)と微量分光光度計(DeNovix社、 DS-11+)を用いてTxn1の活性測定を行なった。
本実験例では、実施例1で作製したHomo、Hetero、および、野生型WTラットについて、寿命を確認した。WTの寿命が2〜3年であるのに比べ、Homoは生後約3-4ヶ月で死亡し、Heteroは9ヶ月頃から死亡する傾向が認められ、Txn1遺伝子改変ラットは寿命が短いこと、また性差があり雄のTxn1遺伝子改変ラットは雌よりも寿命が短いことが確認された(図6)。これにより、当該Txn1遺伝子改変ラットは老化および腎臓病、心血管疾患、高血圧、大動脈解離、慢性閉塞性肺疾患、年齢依存性てんかん、脂質代謝異常、貧血、骨粗鬆症、免疫異常の疾患の性差の研究に有用な動物モデルとして利用可能であると考えられた。
本実験例では、実施例1で作製したHomo、Hetero、および、野生型WTラットの体重を0-12ヶ月までの月齢を追って測定し、健康状態を観察した。図7Aは雄の体重変化を、図7Bは雌の体重変化を示したグラフである。ホモで3ヶ月以降、ヘテロで11ヶ月以降に体重減少が認められる。図7Cに生後4ヶ月時の外見と肉眼的臓器所見を示す。ホモでは顕著なやせ、脊椎変形、脱毛、内臓脂肪の著減、胸腺退縮が認められる。図7Dに生後11ヶ月時の外見と肉眼的臓器所見を示す。ヘテロでは顕著なやせ、脊椎変形、脱毛、内臓脂肪の著減、胸腺退縮が認められる。
本実験例では、実施例1で作製したHomo、Hetero、および、野生型WTラットにおける進行性腎機能障害、高コレステロール血症、貧血の病態について確認した。0-12ヶ月までの月齢を追って血液および尿を採取し、生化学検査を行った。図8のAは血清アルブミン、Bは血中尿素窒素(Blood Urea Nitrogen;Bun)、Cは総コレステロール(Total Cholesterol; T-Cho)、Dはヘモグロビン(Hb)、Eは尿中アルブミン量(Urinary Albumin)、Fは尿潜血、Gは血糖値(Glucose, Glu)、Hは白血球数の月齢推移を示している。Homoは生後3ヶ月で尿中アルブミンとBunが高値となり、末期腎不全にて3-4ヶ月で死亡し、Heteroは7ヶ月頃から尿中アルブミンとBunが上昇し、約1年で末期腎不全となり死亡した。
本実験例では、実施例1で作製したHomo、Hetero、および、野生型WTラットにおける心疾患の病態について確認した。
本実験例では、実施例1で作製したHomo、Hetero、および、野生型WTにおける慢性閉塞性肺疾患の病変について確認した。
本実験例では、実施例1で作製したHomo、Hetero、および、野生型WTにおける胸腺萎縮について確認した。胸腺はT細胞の分化、成熟など免疫系に関与する一次リンパ器官であり、胸小葉とよばれる二葉からなる。成熟した胸腺において外側の部分である皮質には未熟なリンパ球が存在し、内側の髄質はリンパ球のほかにマクロファージや樹状細胞といった抗原提示の細胞などが認められる。
本実験例では、実施例1で作製したHomo、および、野生型WTにおける卵巣萎縮について確認した。WTに比べて、Homoラットでは卵巣萎縮、成熟卵胞の減少が観察された(図14)。
本実験例では、実施例1で作製したHomo、Hetero、および、野生型WTラットについて0-12ヶ月までの月齢を追って採血し、カルシウム、無機リン、アルカリホスファターゼについて生化学検査を行った。また、Homo、Hetero、および、WTについて月齢4ヶ月でコンピュータ断層撮影(Computed Tomography:CT)を行った。
本実験例では、実施例1で作製したHomo、Hetero、および、野生型WTラットにおけるてんかん発作について確認した。てんかん発作は生後4-5週齢に集中して出現し、6週齢以降、てんかん発作は徐々に減少して消失した。より詳しくは、年齢依存的に変容を示すてんかん発作について確認した。
Txn1遺伝子改変ラットのてんかん発作が観察される前の生後3週齢から、4週齢、5週齢、7週齢の脳病変をHE染色で病理検査を行った。ヘテロで3週齢から5週齢において脳の下丘、視床、視床下部に空胞変性を認めた(図17B)。この所見は、Homoでも認められ、より病変部位が広かった。(図17C)に下丘の空胞変性の拡大写真をしめす。この変性は、生後7週齢では治癒した(図17Aと図17B)。
CRISPR/Cas9システムを用いてTxn1遺伝子改変マウスを作成した。まず、マウスTxn1遺伝子の第54番目のコドン近辺の配列を含むガイドRNAを作成し、Cas9蛋白、および、54番目コドンの変異型配列を持つ1本鎖DNA断片(サイレント変異2箇所を含む)と共にC57BL/6J マウスの受精卵に導入した。導入した受精卵136個を仮母親の子宮に移植した。出生した仔マウスF0は9匹であったが5匹が死亡した。残り4匹(F0-#1、F0-#2、F0-#3、F0-#4)の尻尾先端を切除し、得られたDNAを用いて、Txn1遺伝子第54番目のコドンを含む363塩基をPCRで増幅しシークエンス解析を行った。上記の遺伝子改変マウスF0をC57BL/6J野生型マウスと交配を行い、産仔マウスF1を得た。
Claims (2)
- チオレドキシンをコードする遺伝子が改変された非ヒトモデル動物であって、
前記改変された遺伝子が、基質タンパク質のジスルフィド結合を還元するために消費される単位時間あたりのNADPH(ニコチンアミドアデニンジヌクレオチドリン酸)量を減少させ、その減少割合が10%から90%の範囲にあることを特徴とする、非ヒトモデル動物。 - 老化、腎臓病、心血管疾患、高血圧、大動脈解離、慢性閉塞性肺疾患、年齢依存性てんかん、脂質代謝異常、貧血、骨粗鬆症および免疫異常からなる群から選択されるいずれかの疾患にモデルとして適応される、請求項1に記載の非ヒトモデル動物。
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