JP2010051277A - サイトグロビン遺伝子の機能が欠損している非ヒト疾患モデル動物 - Google Patents
サイトグロビン遺伝子の機能が欠損している非ヒト疾患モデル動物 Download PDFInfo
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Abstract
【解決手段】Cygb遺伝子の機能が欠損している非ヒト疾患モデル動物を作製する。当該非ヒト疾患モデル動物を、鉄代謝異常に関連する疾患のモデル動物として使用する。
【選択図】なし
Description
Pinzani M, Vizzutti F, Arena U, Marra F. Technology Insight: noninvasive assessment of liver fibrosis by biochemical scores and elastography. Nat Clin Pract Gastroenterol Hepatol. 2008;5:95-106 Okuyama H, Shimahara Y, Kawada N. The hepatic stellate cell in the post-genomic era. Histol Histopathol. 2002;17:487-495. Knook DL, Blansjaar N, Sleyster EC. Isolation and characterization of Kupfferand endothelial cells from the rat liver. Exp Cell Res. 1977;109:317-29. Wake K. Perisinusoidal stellate cells (fat-storing cells, interstitial cells, lipocytes), their related structure in and around the liver sinusoids, and vitamin A-storing cells in extrahepaticorgans. Int Rev Cytol. 1980;66:303-353. Kawada N, Tran-Thi TA, Klein H, Decker K. The contraction of hepatic stellate (Ito) cells stimulated with vasoactive substances. Possible involvement of endothelin 1 and nitric oxide in the regulation of the sinusoidal tonus. Eur J Biochem. 1993;213:815-823. Friedman SL, Roll FJ, Boyles J, Bissell DM. Hepatic lipocytes: the principal collagen-producing cells of normal rat liver. Proc Natl Acad Sci U S A. 1985;82:8681-8685. Schafer S, Zerbe O, Gressner AM. The synthesis of proteoglycansin fat-storing cells of rat liver. Hepatology. 1987;7:680-687. Friedman SL. Seminars in medicine of the Beth Israel Hospital, Boston. The cellular basis of hepatic fibrosis. Mechanisms and treatment strategies. N Engl J Med. 1993;328:1828-1835. Pinzani M, Gentilini A, Caligiuri A, De Franco R, Pellegrini G, Milani S, Marra F, Gentilini P. Transforming growth factor-beta 1 regulates platelet-derived growth factor receptor beta subunit in human liver fat-storing cells. Hepatology. 1995;21:232-239. Kristensen DB, Kawada N, Imamura K, Miyamoto Y, Tateno C, Seki S, Kuroki T, Yoshizato K. Proteome analysis of rat hepatic stellate cells. Hepatology. 2000;32:268-277. Kawada N, Kristensen DB, Asahina K, Nakatani K, Minamiyama Y, Seki S, Yoshizato K. Characterization of a stellate cell activation-associated protein (STAP) with peroxidaseactivity found in rat hepatic stellate cells. J Biol Chem. 2001;276:25318-25323. Burmester T, Ebner B, Weich B, Hankeln T. Cytoglobin: a novel globin type ubiquitously expressed in vertebrate tissues. Mol Biol Evol. 2002;19:416-421. Nakatani K, Okuyama H, Shimahara Y, Saeki S, Kim DH, Nakajima Y, Seki S, Kawada N, Yoshizato K. Cytoglobin/STAP, its unique localization in splanchnic fibroblast-like cells and function in organ fibrogenesis. Lab Invest. 2004;84:91-101. Sawai H, Kawada N, Yoshizato K, Nakajima H, Aono S, Shiro Y. Characterization of the heme environmental structure of cytoglobin, a fourth globin in humans. Biochemistry. 2003;42:5133-5142. Sugimoto H, Makino M, Sawai H, Kawada N, Yoshizato K, Shiro Y. Structural basis of human cytoglobin for ligandbinding. J Mol Biol. 2004;339:873-885.
項1. サイトグロビン遺伝子の機能が欠損していることを特徴とする、非ヒト疾患モデル動物。
項2. 鉄代謝異常に関連する疾患のモデル動物である、項1に記載の非ヒト疾患モデル動物。
項3. 齧歯目動物である、項1又は2に記載の非ヒト疾患モデル動物。
項4. マウスである、項1乃至3のいずれかに記載の非ヒト疾患モデル動物。
項5. 項1乃至4のいずれかに記載の非ヒト疾患モデル動物に被験物質を投与し、鉄代謝異常に関連する疾患の発症率及び重症度を評価することを特徴とする、鉄代謝異常に関連する疾患の予防又は治療薬のスクリーニング方法。
項6. 被験物質が、サイトグロビンに対するアンタゴニスト又はアゴニストである、項5に記載のスクリーニング方法。
本発明の非ヒト疾患モデル動物は、Cygb遺伝子の機能が欠損しているノックアウト非ヒト動物である。
本発明の非ヒト疾患モデル動物の製造方法については特に制限されず、通常のノックアウト動物の製造方法と同様に実施されるが、一例としてCygb遺伝子が不活性化された胚幹細胞を胚盤胞に導入して得られるキメラ胚を、対象動物の雌の子宮に着床させることにより、キメラ動物を得る方法が例示される。より具体的には、以下に説明する工程(1)〜(6)を経ることにより、本発明の非ヒト疾患モデル動物を得ることができる。
先ず、CygbのゲノムDNAのフラグメントをクローニングする(工程(1))。
次いで、前記工程(1)で得られたフラグメントを改変して、Cygb遺伝子の機能を欠損させる改変フラグメントを作製する(工程(2))。具体的には、前記工程(1)で得られたフラグメントに対して、欠失、置換又は付加を行うことにより、Cygb遺伝子を不活性化する改変フラグメントを得ることができる。前記工程(1)で得られたフラグメントにおいて改変がなされる部位には、Cygb遺伝子のコード領域、好ましくはエクソン1又は2が含まれていることが望ましい。
次いで、前記工程(2)で得られた改変フラグメントが組み込まれたターゲッティングベクターを作製する(工程(3))。
次いで、前記工程(3)で得られたターゲッティングベクターを胚性幹細胞に相同性組み換えを行うことにより、Cygb遺伝子の機能が欠損した胚性幹細胞を作製する(工程(4))。
次いで、前記工程(4)で得られたCygb遺伝子の機能が欠損した胚性幹細胞が導入されたキメラ胚を作製する(工程(5))。
次いで、前記工程(5)で得られたキメラ胚を雌非ヒト動物の子宮に着床させて、キメラ動物を発生させる(工程(6))。
前記工程(6)で得られたキメラ動物同士を交配させることにより、ヘテロ接合体のCygb遺伝子欠損非ヒト動物、又はホモ接合体のCygb遺伝子欠損非ヒト動物を得ることができる。得られたCygb遺伝子欠損非ヒト動物が、ヘテロ接合体であるか、或いはホモ接合体であるかについては、サザンブロット法等の公知の解析手法によって確認できる。
実施例1 Cygb遺伝子欠損マウスの作製及び確認
(1) Cygb遺伝子欠損マウスの作製
マウス129SvゲノムDNAから、PCR法によりCygb遺伝子の5′上流域の5.9kbDNAフラグメントをクローニングした。このフラグメントは、CygbゲノムDNAの5’UTRおよびエクソン1の一部を有する。さらに、マウス129SvゲノムDNAから、PCR法によりCygb遺伝子の3′下流域の6.9kbDNAフラグメントをクローニングした。このフラグメントは、サイトグロビンゲノムDNAのエクソン3,4および3’UTRを有する。上流5.9kbDNAフラグメントの下流にホスホグリセリン酸キナーゼプロモーター/ネオマイシン抵抗遺伝子カセット(PGK−neo)を結合し、さらにその下流に、6.9kbのDNAフラグメントを結合した。ネオマイシン抵抗遺伝子カセットは、両側をバクテリオファージP1のloxPとよばれる部位特異的組換え配列で挟まれている。この改変フラグメントを、プラスミドベクターpBluescript II(ストラタジーン株式会社)に組み込んで、ターゲッティングベクターpTVFloxPGKneo/Cg#3を得た。
正しく相同組換えしたES細胞を、BDF1マウスの交配から得られた嚢胚と凝集させ、偽妊娠マウスの子宮に移植し、キメラ胚を得た。得られたキメラ胚を、C57BL/6Jマウスと交配して、Cygb遺伝子がヘテロのマウスを得た。このようなCygb遺伝子をヘテロにもつ雄雌マウス同士を交配させ、Cygb遺伝子を欠失したホモマウス(Cygb遺伝子欠損マウス)を得た。
上記で作製されたマウス尾からDNAを抽出し、PCRを行いCygb遺伝子とネオマイシン遺伝子の存在をプライマー[CTCCCAGCCGGGACCGCGGTGGCCTT (両者に共通するforward primer;配列番号1), GGAGCCGAGGCCGGTGCGTGCGAGGC (Cygbに対するreverse primer;配列番号2), GTGGGGTGGGATTAGATAAATGCCTGCTCT (Neoに対するreverse primer;配列番号3)]で検討したところ、野生型ではCygb+/Neo-、ヘテロマウスではCygb+/Neo+であり、ホモマウスでは完全にCygb 遺伝子が消失していた(図2参照)。更に、上記で作製されたマウスの肝臓をホモゲネートし、その10μgをSDS-PAGEで分離後、抗ラットCygbペプチド抗体(Kawada N, KristensenDB, et al. J Biol Chem. 2001;276:25318)を用いてウエスタンブロット法でCygb蛋白発現を確認したところ、ヘテロマウスではCygb蛋白質が半分に減少し、ホモマウスでは完全にCygb蛋白質が消失していることを確認した(図3参照)。
上記実施例1で得られたCygb遺伝子欠損マウス(ホモマウス)の特性を評価するために、生育及び生理学的特徴を経時的に観察した。
Cygb遺伝子欠損マウスは野生型マウスの産子数(n=8.8±0.97)に比し、一回の出産により生まれる産子数(n=4.4±1.95)が有意に少ないことが判明した。また生後2週間以内に死亡に至る数はn=1.3±1.41であり繁殖率に差が生じた。その後は野生型とほぼ同様に成長し、寿命に明らかな差は認められなかった。
10週齢のCygb遺伝子欠損マウスでは、脾臓に軽度腫大が見られた。体重並びに臓器重量を測定すると野生型マウスにおいては、体重:25.88±0.72g(n=5)、肝重量:1.26±0.04g(n=5)、脾臓重量:0.064±0.059g(n=5)であったのに対し、Cygb遺伝子欠損マウスでは体重:30.34±1.38g(n=5)(p<0.01)、肝重量:1.50±0.11g(n=5)(p<0.05)、脾臓重量:0.089±0.017g(n=5)(p<0.01)であり、両群の重量差には有意差が認められた。当該脾臓について更に詳細に検討するために、これを4%パラホルムアルデヒドを用いて固定してパラフィン包埋した後に、4μmに薄切して脱パラフィンした組織を用いて各種染色を行った。顕微鏡下に観察すると、ベルリン青染色で赤脾髄に沿って鉄の沈着がみられることが判明し、赤脾髄に変化が生じることが明らかになった(図6参照)。
10ヶ月齢のマウスを開腹すると、野生型マウスに比較してCygb遺伝子欠損マウスでは肝臓と脾臓が腫大していた(図9参照)。野生型マウスの肝臓が1.55±0.23g(n=5)であったのに対して、Cygb-/-マウスでは1.74±0.06g(n=5)であった。さらに脾臓は野生型0.11±0.027g(n=5)に対しCygb遺伝子欠損マウス0.16±0.046g(n=5)(p<0.01)と有意な腫大が認められた。
Claims (6)
- サイトグロビン遺伝子の機能が欠損していることを特徴とする、非ヒト疾患モデル動物。
- 鉄代謝異常に関連する疾患のモデル動物である、請求項1に記載の非ヒト疾患モデル動物。
- 齧歯目動物である、請求項1又は2に記載の非ヒト疾患モデル動物。
- マウスである、請求項1乃至3のいずれかに記載の非ヒト疾患モデル動物。
- 請求項1乃至4のいずれかに記載の非ヒト疾患モデル動物に被験物質を投与し、鉄代謝異常に関連する疾患の発症率及び重症度を評価することを特徴とする、鉄代謝異常に関連する疾患の予防又は治療薬のスクリーニング方法。
- 被験物質が、サイトグロビンに対するアンタゴニスト又はアゴニストである、請求項5に記載のスクリーニング方法。
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RU2610536C1 (ru) * | 2015-10-20 | 2017-02-13 | Федеральное государственное бюджетное научное учреждение "Научно-исследовательский институт акушерства, гинекологии и репродуктологии имени Д.О. Отта" | Способ моделирования острого жирового гепатоза беременных |
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