JP4230911B2 - 幹細胞由来のヒト病態モデルとしての遺伝子導入非ヒト哺乳動物 - Google Patents
幹細胞由来のヒト病態モデルとしての遺伝子導入非ヒト哺乳動物 Download PDFInfo
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Description
(i)本発明のDNA構造体を遺伝子導入非ヒト哺乳動物の受精卵母細胞に導入すること。
(ii)子孫を生成するため前記受精卵母細胞を偽妊娠の乳母に移植すること。
(iii)幹細胞由来のヒト病態に関与する染色体異常によって形成された遺伝子および/または活性化遺伝子の存在を評価するために前記子孫を分析すること。
(i)Sca−1+ 細胞に遺伝子の発現を導くプロモータと治療および/または予防を行う幹細胞由来のヒト病体に関連する染色体異常の治療および/または予防のための適切な治療遺伝子とから成るDNA構造体を調製すること(この場合に、前記治療遺伝子はSca−1+ 細胞に発現を導く遺伝子の制御下にある)、および(ii)細胞に外因性遺伝子を導入するプロセスにおいて遺伝子が正常に機能するように細胞内の伝達およびのその生物学的利用能を促進するベクターまたはシステムにDNA構造体を組み入れること。実例として、前記ベクターまたはシステムは、例えば、アデノウィルス、レンチウィルスなどのウィルス性ベクターや、DNA−リポソーム、DNA−ポリマー、DNA−ポリマーリポソーム複合体などの非ウィル性のものが挙げられる(ファング,ハングおよびワグナー編集、アカデミック プレス(1999年)発行の「遺伝子治療用の非ウイルス性ベクター」「Nonviral Vectors for Gene Therapy」edited by Huang,Hung,Wagner,「Academic Press(1999)」を参照のこと)。
[実施例]
プロモータ:C57BL76マウス(Jackson Laboratory)において異なる導入遺伝子(cDNA)の組織特有の発現を導くためにプロモータpLy−6E.1が使用された。このプロモータは優れた特徴があり、使用された16キロベース(KB)の断片には、Sca−1+ 細胞における選択的な発現に必要な全ての要素が含まれている(マイルス シ,サンチス エム ジェイ,シンクレアー エイおよびジェルサック著、(1997年)、成人造血幹細胞におけるLy−6E1(Sca−1)導入遺伝子の発現およびマウス胎芽の発育、発育、第124巻、第537頁乃至第547頁「Miles C,Sanchez M−J,Sinclair A,and Dzierzak,E (1997)「Expression of the Ly−6E1(Sca−1)transgene in adult haematopoietic stem cells and the developing mouse embryo」Development 124,537,547」)。
遺伝子:使用された遺伝子は以下の通り。
ヒトHOX11−T細胞急性リンパ母細胞性白血病に関連する異常染色体によって活性化される遺伝子。
これらの動物は市販されており、例えば、Jackson Laboratory(米国)から入手できる。
遺伝子導入マウスの発生および選択(スクリーニング)
各種のcDNA(ヒトBCR−ABLP 1 9 0 、ヒトBCR−ABLP 2 1 0 、マウスSlug、マウスSnail、HOX11、ヒトLMO2/RHOM2およびマウスTAL1)を、pLy−6E.1プロモータのClaIサイトにおいてクローン化した。適切な制限エンドヌクレアーゼでの消化により異なるcDNAが得られた。各cDNAは、従来技術によりClaIで消化されたpLy−6E.1プロモータを含むベクターにおいてクローン化された(分子クローン化、第3版、サムブロークおよびルーセル著、シーエスエッチエルプレス(2001年)発行「Molecular Cloning,third edition,CSHL Press by Sambrook,and Russell(2001年)」)。
血球計算(サイトメトリー)のための染色には、モノクローナル抗マウス抗体(CD45R/B220,Thy−1.1およびThy−1.2、骨髄性マーカ(Mac 1/CD11bおよびGr−1)およびSig)をフィコエリトリン(PE)と結合させる(すべてPharmingen製)。ルーチン技術によって得られた全血液サンプルからの細胞の懸濁液はFcによるレセプターとの結合をブロックするために精製されたCD32/CD16抗マウス(Pharmingen)を用いて、それぞれ室温において4℃に適切に希釈された各種の抗体で培養された。赤血球は溶解液(Becton Dickinson)を用いて溶解した。サンプルはリン酸緩衝食塩水(PBS)で2度洗浄し、そしてPBS中に再懸濁させた。サンプル中の死細胞はプロピジウムヨウ化物(propidium iodide)を用いた染色によって排除した。サンプルおよびデータはCellQuestプログラム(Becton Dickinson)を用いてFACScanで分析した。
mRNAはキメラマウスの異なる組織から得た。cDNAは、RNaseのDNAse I(HT)で処理したRNAの各標本の逆転写によって得た。cDNAは各場合に特別な表示器を用いてPCRにかけた(ダイレクトイニシエーター(5' )は、cDNAの最初の20ベースに対応し、インバースイニシエーター(3' )は、cDNAの最後の20ベースに対して補足的であった)。反応は、Taqポリメラーゼのサプライヤー(Perkin−Elmer Cetus)の指示に従って、95℃で1分、55℃で1分、72℃で1分のサイクルを25サイクル行い、そして最後に72℃で10分延長した。
組織サンプルは、PBSにおいて4%のホルムアルデヒドで固定し、そしてパラフィンに浸した。処理した薄膜を切り出し、ルーチン技術によってヘマトキシリン−エオシンを用いて染色した。薄膜を試験しかつ写真撮影した。
脾臓から採取した細胞の懸濁液を従来の方法を使用する免疫ブロット法で分析した(抗体、研究所手引き、ハーロウおよびレーン著、CSH、1988年発行「Antibodies:A Laboratory manualHarlow and Lane,CSH,1988」)。
DNAは異なる細胞から従来の技術を用いて調製した。DNAはBamHIで分解し、特定の免疫グロブリンのプローブでサザンブロット法によって分析した(血液(迅速出版)第90巻、第2168頁乃至第2174頁、1997年「Blood(rapid publication)90:2168−2174(1997)」)。
ドナーマウスの器官から得た細胞は懸濁され、洗浄され、4〜6か月のレセプターマウスの尾に静脈注射された(NOD/SCDI)(The Jackson Laboratory)。マウスの観察は1週間に1度行われ、死にかけた状態の時に、DNA分析用の組織病理研究および組織収集のために犠牲にされた。
1.1 Sca−1 + BCR−ABL P 2 1 0 遺伝子導入マウスの発生
遺伝子生成物、BCR−ABLP 2 1 0 (t(9:22)(q34;q11)の結果として生じかつ慢性骨髄性白血病に関連する遺伝子融合)の発現の直接的な結果を生体内で確認するため、キメラヒトタンパク質BCR−ABLP 2 1 0 のcDNAをマウスのプロモータpLy−6E.1のコントロール下でクローン化し、受精卵母細胞を前記「方法」の項で述べた方法に従ってC57BL/6J x CBAマウスに注入した。2匹の遺伝子導入始祖マウス(Sca−1+ BCR−ABLP 2 1 0 )が、導入遺伝子を生殖細胞系に伝達する能力を示した。導入遺伝子の発現が両方の系で観察され、F7レベル(第7世代)まで子孫が繁殖された。導入遺伝子の発現は、PCRおよび/またはウェスタンブロット分析法によって証明された。両方の細胞系はSca−1+ 細胞に優先的な発現が認められた。導入遺伝子の発現はオスとメスの両方のマウスにおいて観察され、Sca−1+ BCR−ABLP 2 1 0 遺伝子導入マウスの両方の系で結果は同様であった。
生体内で遺伝子生成物、ヒトBCR−ABLP 1 9 0 、マウスSlug、マウスSnail、ヒトHOX11、ヒトRHOM2/LMO−2およびマウスTAL1の発現の直接的な結果を調べるために、対応するcDNAをマウスのプロモータpLy−6b.1のコントロール下でクローン化し、生じた構造体を前もって直線化しそしてC57BL/6J x CBAマウスの受精卵母細胞に前記「方法」の項で示される方法に従って注入した。各構造体に対して、導入遺伝子を生殖細胞系に伝達する能力を有する2匹の遺伝子導入始祖マウスが得られた。このようにして実験を行い、以下のように認識された遺伝子導入マウスを得た。
Sca−1+ BCR−ABLP l 9 0 (B細胞急性リンパ母細胞性白血病を生じる)(図3参照):
Sca−1+ Slug(造血幹細胞の移動を生じるが、白血病を有しない);
Sca−1+ Snail(造血幹細胞の移動を生じるが、白血病を有しない);
Sca−1+ HOX11(T細胞急性リンパ母細胞性白血病を生じる);
Sca−1+ RHOM2/LMO−2(T細胞急性リンパ母細胞性白血病を生じる);
Sca−1+ TAL1(T細胞急性リンパ母細胞性白血病を生じる);
全ての場合に、両方の系において導入遺伝子の発現が観察され、F7レベル(第7世代)まで子孫が繁殖された。導入遺伝子の発現はPCRおよび/またはウェスタンブロット分析法によって確認された。両方の細胞系はSca−1+ 細胞に優先的な発現が認められた。導入遺伝子の発現はオスとメスの両方のマウスにおいて観察され、両方の系で結果は同様であった。
ヒト病態では、遺伝子のキメラ生成物Sca−1+ BCR−ABLP 2 1 0 、Sca−1+ BCR−ABLP 1 9 0 、Sca−1+ Slug、Sca−1+ Snail、Sca−1+ HOX11、Sca−1+ RHOM2/LMO−2およびSca−1+ TAL1は各種の白血病に、特に、慢性骨髄性白血病(BCR−ABLP 2 1 0 )、B細胞急性リンパ母細胞性白血病(BCR−ABLP 1 9 0 、)およびT細胞急性リンパ母細胞性白血病(HOX11、RHOM2/LMO−2およびTAL1)に関与しているが、適切な細胞タイプの発現を行うためのプロモータの選択が難しいことから、前記白血病のための現在のマウスモデルは、前記病態を一貫して再現することに失敗している(遺伝子学評論年鑑、(1997年)、第31巻、第429頁乃至第453頁および現代遺伝子学(2000年)、第1頁、第70頁乃至第80頁「Annu.Rev.Genetics(1997)31:429−453;Current genomics(2000)、1:71−80」)。
Claims (7)
- 遺伝子導入マウスであって、ヒトBCR−ABLp210、ヒトBCR−ABLp190、ヒトHOX11、ヒトRHOM2/LMO−2およびヒトTAL1から選択される遺伝子を含んでなるDNA構造体をそのゲノムに含み、
ここで当該マウスはSca−1 + BCR−ABL p210 、Sca−1 + BCR−ABL p190 、Sca−1 + HOX11、Sca−1 + RHOM2/LMO−2およびSca−1 + TAL1として確認されているマウスの中から選択され、
ここで当該Sca−1 + BCR−ABL p210 マウスは慢性骨髄性白血病を示し、当該Sca−1 + BCR−ABL p190 マウスはB細胞急性リンパ母細胞性白血病を示し、そして当該Sca−1 + HOX11マウス、Sca−1 + RHOM2/LMO−2マウスおよびSca−1 + TAL1マウスはT細胞急性リンパ母細胞性白血病を示す、
そして、該遺伝子がマウスpLy6E.1プロモータの制御下にある、
前記遺伝子導入マウス。 - 請求項1に記載の遺伝子導入マウスの子孫。
- 請求項1または2に記載の遺伝子導入マウスまたはその子孫から得られる細胞。
- 請求項1に記載の遺伝子導入マウスを調製する方法であって、以下:
(i)DNA構造体をマウスの受精母細胞に導入する、ここで当該DNAはヒトBCR−ABLp210、ヒトBCR−ABLp190、ヒトHOX11、ヒトRHOM2/LMO−2およびヒトTAL1から選択される遺伝子を含み、該遺伝子はマウスpLy6E.1プロモータの制御下にある;
(ii)前記受精卵母細胞を子孫を生成するために偽妊娠の乳母に移植する;および
(iii)前記(i)の存在を評価するために前記子孫を分析する;
を含み、
ここで当該マウスはSca−1 + BCR−ABL p210 、Sca−1 + BCR−ABL p190 、Sca−1 + HOX11、Sca−1 + RHOM2/LMO−2およびSca−1 + TAL1として確認されているマウスの中から選択され、
ここで当該Sca−1 + BCR−ABL p210 マウスは慢性骨髄性白血病を示し、当該Sca−1 + BCR−ABL p190 マウスはB細胞急性リンパ母細胞性白血病を示し、そして当該Sca−1 + HOX11マウス、Sca−1 + RHOM2/LMO−2マウスおよびSca−1 + TAL1マウスはT細胞急性リンパ母細胞性白血病を示す、
前記方法。 - ヒト病態を再現する遺伝子導入マウスを発生させるためのマウスpLy6E.1プロモータの使用であって、当該マウスはヒトBCR−ABLp210、ヒトBCR−ABLp190、ヒトHOX11、ヒトRHOM2/LMO−2およびヒトTAL1から選択される遺伝子を含有し、
ここで当該マウスはSca−1 + BCR−ABL p210 、Sca−1 + BCR−ABL p190 、Sca−1 + HOX11、Sca−1 + RHOM2/LMO−2およびSca−1 + TAL1として確認されているマウスの中から選択され、
ここで当該Sca−1 + BCR−ABL p210 マウスは慢性骨髄性白血病を示し、当該Sca−1 + BCR−ABL p190 マウスはB細胞急性リンパ母細胞性白血病を示し、そして当該Sca−1 + HOX11マウス、Sca−1 + RHOM2/LMO−2マウスおよびSca−1 + TAL1マウスはT細胞急性リンパ母細胞性白血病を示す、
前記使用。 - 造血幹細胞または非造血幹細胞のいずれかに由来する腫瘍性ヒト病態を治療および/または予防するための潜在的に有効な化合物を評価するための請求項1若しくは2に記載の遺伝子導入マウス若しくはその子孫または請求項3に記載の細胞の使用。
- ヒト病態がB細胞急性リンパ母細胞性白血病、T細胞急性リンパ母細胞性白血病および造血幹細胞または胚幹細胞の移動から選択される請求項6に記載の使用。
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ES2301268B1 (es) | 2004-10-25 | 2009-05-01 | Centro De Investigacion Biomolecular Aplicada Salamanca, S.L. | Empleo del gen slug, o de sus productos de replicacion, transcripcion o expresion, en la identificacion, diagnostico, prevencion o tratamiento de la diseminacion del cancer y/o desarrollo de metastasis. |
EP1726208A3 (en) | 2005-05-24 | 2007-02-28 | Centro de Investigacion Biomolecular Aplicada S.L. | Murine stem cells and applications thereof |
WO2008029290A2 (en) * | 2006-09-07 | 2008-03-13 | Universidad De Salamanca | Identification of cancer stem cells using genetic markers |
US20110302665A1 (en) * | 2008-07-02 | 2011-12-08 | Oktay Kirak | Non-human mammals with t or b cells having predefined specificity |
CN108271741B (zh) * | 2018-01-19 | 2020-11-03 | 华北理工大学 | 一种黑腹果蝇GMR>Snail细胞死亡模型的建立方法 |
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WO1991007489A1 (en) * | 1989-11-22 | 1991-05-30 | Childrens Hospital Of Los Angeles | Bcr/abl transgenic animals as models for philadelphia chromosome positive chronic myelogenous and acute lymphoblastic leukemia |
US5928638A (en) * | 1996-06-17 | 1999-07-27 | Systemix, Inc. | Methods for gene transfer |
ES2161612B1 (es) * | 1999-07-01 | 2002-06-01 | Consejo Superior Investigacion | Procedimiento para identificar un compuesto que inhiba la funcion represora de snail. |
ES2185472B1 (es) * | 2001-01-23 | 2004-10-16 | Universidad De Salamanca (O.T.R.I.) | Empleo del gen slug, o de sus productos de transcripcion o expresion, en la deteccion y/o tratamiento de celulas cancerosas. |
ES2195751B1 (es) * | 2001-11-27 | 2005-04-01 | Universidad De Salamanca (Otri) | Mamiferos no humanos transgenicos como modelos para patologias humanas con origen en celulas stem. |
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ES2311636T3 (es) | 2009-02-16 |
PT1449920E (pt) | 2008-11-13 |
DK1449920T3 (da) | 2008-12-01 |
ES2195751B1 (es) | 2005-04-01 |
JP2009055910A (ja) | 2009-03-19 |
US20060130169A1 (en) | 2006-06-15 |
US7456333B2 (en) | 2008-11-25 |
US20060130168A1 (en) | 2006-06-15 |
CA2466372C (en) | 2010-02-09 |
CY1108473T1 (el) | 2014-04-09 |
US20060130166A1 (en) | 2006-06-15 |
US20060130167A1 (en) | 2006-06-15 |
ATE403723T1 (de) | 2008-08-15 |
DE60228117D1 (de) | 2008-09-18 |
EP1449920A1 (en) | 2004-08-25 |
AU2002350745A1 (en) | 2003-06-10 |
US20050108784A1 (en) | 2005-05-19 |
ES2195751A1 (es) | 2003-12-01 |
US7626074B2 (en) | 2009-12-01 |
EP1449920B1 (en) | 2008-08-06 |
CA2466372A1 (en) | 2003-06-05 |
WO2003046181A1 (es) | 2003-06-05 |
EP2223939A1 (en) | 2010-09-01 |
JP4504444B2 (ja) | 2010-07-14 |
JP2005510241A (ja) | 2005-04-21 |
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