JP6216456B2 - 組換え活性遺伝子2遺伝子ターゲッティングベクター、そのベクターが導入された兔疫細胞欠乏形質転換ミニクローンブタの生産とその製造方法および活用 - Google Patents
組換え活性遺伝子2遺伝子ターゲッティングベクター、そのベクターが導入された兔疫細胞欠乏形質転換ミニクローンブタの生産とその製造方法および活用 Download PDFInfo
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Description
本発明の他の実施形態において、前記RFPおよびGFPの発現は、流動細胞分析法によって検出することが好ましいが、これに限定されない。
遺伝子ターゲッティングのために、2−3百万の細胞を、レポーターベクターを有するTALEN構築物でトランスフェクションさせた;2μgの各構築物/百万細胞あたり。それら細胞をBTX Electro Cell Manipulator(Harvard Apparatus、Holliston、MA)を用いて、490V、1msec、3パルスでその構築物で電気穿孔した。その後、48時間、T75フラスコでプレートした細胞を、Beckman Coulter MoFlo XDPを用いてGTPポジティブ細胞に対して分類した。その分類された細胞を96−ウェルプレートでプレートした。10日後、半分の細胞を遺伝子型のために使用した。TALENSを挿入した後、indelの存在を調べるために、TALEN切断部位周辺のゲノムDNAの切片をPCRで増幅した。培養細胞由来ゲノムDNAを細胞破砕バッファーを用いて分離した後、そのゲノムDNAをPCRに使用した。増幅のためのPCR条件は、95℃で2分の初期変性、32サイクルの変性のための94℃で30秒、アニーリングのための55℃で30秒、拡張のための72℃で30分を伴った(表1のPCRプライマーセットについては参照)。PCR産物の予測された大きさは、IL2RGに対しては417bpであり、RAG2に対しては426bpであった。そのPCR産物を、indelの存在を同定するためにシーケンシングした。
SCNT胚芽を生産するために、雌ブタ由来卵母細胞(oocytes)をART(Madison、WI)から購入した。その卵母細胞を成熟培地(TCM199 with 2.9mM Hepes、5μg/ml insulin、10ng/ml EGF、0.5μg/ml p−FSH、0.91mM pyruvate、0.5mM cysteine、10%ブタ卵胞液、25ng/ml gentamicin)でオーバーナイト輸送して、24時間後に新鮮培地にトランスファーした。40−42時間の成熟後、卵丘細胞(cumulus cells)を0.1%hyaluronidaseの存在下でボルテキシングして、その卵母細胞から除去した。操作の間、卵母細胞を7.0μg/ml cytochalasin Bが補充された操作培地に位置させた。中期II板(metaphase II plate)を含む隣接した原形質部分に沿った極体を除去し、供与細胞を薄いガラス毛細管を用いて囲卵腔(perivitelline space)に位置させた。
IHCのために、10%ホルマリンを有する中性バッファーで固定化された組織を使用した。その組織をIHCのためにスライド上で充填した。内生的なperoxidase活性を先に3%hydrogen peroxidaseで抑制した。サンプルをBorg Decloakerで前処理した後、背景Sniper溶液でブロッキングした。洗浄後、サンプルをB細胞(CD79A;Diagnostic Biosystems−#Mob118、1:100)またはT細胞(CD3;DAKO−#A0452、1:400)に特異的な1次抗体で培養した。培養後、サンプルを洗浄し、HRP付着した2次抗体で培養した。その後、サンプルを、その信号を見るために、Romulin Red Chromogensで染色した。また、サンプルを、背景を提供するために、IP FLX Hematoxylinで染色した。The Borg、Sniper、Romulin RedおよびIP FLX hematoxylinはいずれもBiocare(Concord、CA)から購入した。すべての顕微鏡写真はOlympus DP70高解像デジタル顕微鏡カメラ(Olympus、Center Valley、PA)が具備されたZeiss Axiophot顕微鏡(Carl Zeiss、Oberkochen、Germany)を用いて得た。
安楽死させた野生型および二重対立遺伝子型コブタ由来脾臓の部分を10%ウシ胎児血清で補充されたRPMI−1640培地(Mediatech、Inc.、Manassas、VA)に集めて、メス刃で研ぎ、20ga.針を通して数回吸入し、70mのナイロンメッシュ細胞ストレーナー(BD Biosciences、San Jose、CA)を通して押出した。その後、脾臓細胞懸濁液を、赤血球を破砕するために、Pharm Lyse溶液(BD Biosciences)で15分培養した後、5分間200×gでペレット化した。その上澄液を捨てた後、そのペレットを冷蔵染色バッファー(BD Pharmingen)に再富化し、細胞をヘマサイトメーターで計数した。その後、細胞を200μLの染色バッファーに5×106細胞のアリコートに分けた。FITC−付着したマウスanti−pig CD21、マウスanti−pig CD3ε、およびマウスanti T−2マイコトキシンIgG1k(isotype対照群)(SouthernBiotech、Birmingham、AL)を細胞に0.5μg/1×106細胞として添加し、暗条件下、4℃で培養した。その後、細胞を2回洗浄し、新鮮な染色バッファーに再富化した。細胞を、ミズーリ大学のCell and Immunobiology Core施設でCyAn ADP流動細胞分析器(Beckman Coulter、Brea、CA)を用いて分析した。データを、Summit v4.3ソフトウェア(Beckman Coulter)を用いて分析した。
本発明で使用されたTALENs由来の推定オフターゲット配列を同定するために、バイオインフォマティクス方法を、最近、ブタゲノムアセンブリ(Sscrofa10.2)由来の各TALEN結合部位に類似の配列を同定するのに使用した。PCRプライマーをヌクレオチド差の数に基づく隣接した最も類似のオフターゲット部位を考案した。この部位を基盤(founder)動物で増幅し、Surveyor nucleaseアッセイを用いてオフ−ターゲッティングイベントのテストを行った(表2および3)。PCR増幅後、300−500ngのPCR産物(10−15μl)を新鮮なチューブに伝達し、thermocyclerプログラム:(95℃、2分、95℃から85℃まで秒あたり−2℃ずつ、85℃から25℃まで秒あたり−0.1℃、無限定的に4℃)に従って変性しリアニーリングした。1μlのSurveyor nucleaseと1μlのSurveyorエンハンサーを添加し、42℃で30分間培養した。その後、その反応を氷上に直ちに位置させ、6XSurveyor nuclease中止バッファーと6X染料をその反応に添加した。そのサンプルを2.0%アガロースゲル上で電気泳動した。
組織を0.01M PBS(pH7.4)に溶解した4%(w/v)パラホルムアルデヒドで固定し、PBSで洗浄し、エタノール(70%、90%、および100%)で脱水し、パラフィンワックスに包埋した。その切片(6μm)を再水和(キシレン5分;エタノール100%、95%、70%、それぞれ2分)し、TUNEL染色前に蒸留水で洗浄した。その切片を室温でプロテイナーゼK(10mM Tris/HCl、pH7.5で20μg/ml)とともに、30分間培養した。切片をTUNELミックスを具備した湿ったチャンバ(In situ Cell Death Detection kit、Roche、Swiss)にて室温で10分間培養した。3回のPBS洗浄後、スライドをDAPIを具備したVECTASHIELD Mounting Media(VECTOR、USA)に装着した。
野生型、RAG2単一対立形質、およびRAG2二重対立形質ブタ由来の細胞の増殖を、24時間および48時間後、培養物内の細胞数をカウントして測定した。ラミニンコーティングされた12−ウェル(well)プレートに細胞を1x104細胞/ウェルで接種した。0、24および48時間目に、それぞれのウェルの細胞数をカウントした。細胞を0.4%トリパンブルー染料(Bio−Rad)で染色して生存率を確認した。それぞれの時点で細胞の数を、TC10自動細胞カウンター(Bio−Rad)を用いて測定したが、それぞれのブタ由来の3つの独立した試料が使用された。24および48時間目の細胞数の差を、統計学的分析システム(SAS Institute、Cary、NC)を用いて比較した。
ヒト臍帯組織を大学病院(University of Missouri、Columbia、MO)から新鮮で無菌的に採取した。組織採取(プロジェクト#1201132)はミズーリ大学の健康科学倫理委員会の承認を受けた。その組織をリン酸塩緩衝塩水(PBS)で2回以上洗浄して血液細胞を除去し、DMEM培地でハサミを用いて1?mm3の断片に細かく粉砕して、付着細胞を外植方法(explants method)により伝達した。その断片化された組織を、10%FBS、1%非必須アミノ酸、2mMグルタミン、0.1mM2−メルカプトエタノール、および4ng/ml FGF2を含有したDMEM培地(Thermo)で0.1%ゼラチンコーティングされた48−ウェルプレート(ウェルあたり1切れ)に位置させた後、4%O2/5%CO2/91%N2の湿った雰囲気を含有する培養器にて37℃で培養した。その培養物を最初5−7日間撹乱しない状態で維持し、Primocin(InvivoGen、San Diego、CA)を添加して、1次培養で細菌および真菌成長の危険を減少させた。次に、Primocinまたは他の抗生剤がない培地を、組織断片由来の線維細胞性付着細胞がウェル内で成長するまで2日ごとに補充した。その線維芽細胞成長は1週の培養後粉砕された組織の周辺で始まった。10−11日目に、その線維芽細胞をTrypLE(Invitrogen)を用いて48−ウェルプレートからT25フラスコに移した。その細胞は、フラスコで約14日目にコンフルエンス(confluence)に到達し、iPSCへのリプログラミングのために膨張した。
通常のリプログラミング遺伝子であるPOU5F1、SOX2、KLF4、およびLIN−28以外にも、p53抑制および非形質転換L−MYCのためのshRNAを有するエピソームベクターを用いてOkitaらが開発したプロトコルを用いて線維芽細胞をリプログラミングした。3μgのエピソームプラスミドのY4結合体を、Nucleofector II機器(Lonza、Basel、Switzerland)およびAmaxa NHDF Nucleofectorキット(Lonza)を用いて、製造会社の指示に従って、6x105細胞内に電気穿孔により導入した。その機器内の電気穿孔プログラムである「U−020」を利用した。その細胞を、前記条件での培養により2〜4日間回復させた。細胞(2x105)をMatrigel(BD Bioscience、San Jose、CA)コーティングされた100mmディッシュ(dish)に位置させた。翌日、その培養培地をmTeSR1(Stem Cell Technologies、Vancouver、Canada)に入れ替えた。ヒトESCと類似のコロニーが形質導入後約14日目に現れ、そのコロニーを、約20日目に機械的に分離して、Matrigel上で無支持細胞培養(feeder−free condition)した。
デジタルカメラCoolpix5000(Nikon、Melville、NY)を具備したOlympus CKX41倒立顕微鏡を用いてiPSCのイメージを取得した。免疫蛍光分析のために、Matrigelコーティングされたカバースリップ上で細胞を成長させた。4%パラホルムアルデヒド/PBSで10分間固定し、1.0%Triton X−100/PBSで30分間透過(permeabilization)した後、カバースリップをPBSに溶解した5%ヤギ血清/5%BSAに1時間位置させた。次に、その細胞を適切に希釈した抗体であるPOU5F1(1:200、sc−5279、Santa Cruz Biotechnology)、NANOG(1:100、ab109250、Abcam)、およびSSEA4(1:100、#4755p、Cell Signaling Technology)とともに、4℃で一晩培養した後、Alexa Fluor568または488−標識ヤギ抗−マウスまたはウサギ抗体(1:500)とともに培養した。ORCA−AG CCDカメラを具備したOlympus IX70倒立顕微鏡(http://www.biotech.missouri.edu/mcc/Olympus.html)を用いてイメージを取得した。
1日目に2つの個体由来の2つのヒトiPSC細胞株(4〜9の継代数)を25%Matrigel溶液とともに、0.2mlの体積で5匹のブタに皮下注射(部位あたり5百万または1000万個の細胞)したが、その5匹のうち3匹は二重対立形質RAG2変形を有し、2匹は対照群としてRAG2の単一対立形質変形を有した。その培養されたiPSC細胞をディスパーゼ(StemCell Technologies)およびスクレイピングにより剥がした。遠心分離(200xg、5min)後、細胞ペレットを0.1mlのmTeSR1培地に再懸濁し、同一体積の50%Matrigelと混合した。次に、その細胞を氷上で冷やし、1ml注射器(BD、Franklin Lakes、NJ)にローディングした後、22ゲージニードルを通してブタ1匹あたり2つの部位に注射したが、1つの部位は耳で、他の1つは脇腹であった。次に、形成される腫瘍を切断除去して、10%(v/v)中性緩衝ホルマリンに固定した。パラフィン包埋された組織を切片にした後、ヘマトキシリンおよびエオシン(H&E)で染色した。ブタのiPSC(iTR)から生成された栄養胚葉(trophoblast)表現型を発現するブタ細胞を二重対立形質RAG2突然変異ブタのうちの1匹に移植した。iTRサブタイプ細胞株(p29)の千万個の細胞をTrypLEおよびスクレイピングによって剥がした。細胞懸濁液をヒトiPSCとして製造して、左耳に皮下注射した。その細胞移植過程はブラインド方式で行ったが、その過程を行う個体はブタの遺伝的状態を知らなかった。
免疫組織化学的(IHC)分析のために、組織を中性緩衝液(Fisher、99−909−14)に溶解した10%ホルマリンに固定し、パリピンに包埋し、ガラススライド上で切片(5μm)を作った。まず、その組織切片を3%水素ペルオキシダーゼで1時間処理して、内因性ペルオキシダーゼ活性を遮断した。次に、その試料を抗原復旧用Borg Decloaker(Biocare Medical、CA)溶液で前処理した後、Background Sniper(Biocare Medical、CA)溶液でブロッキングした。洗浄後、試料を1次抗体とともに培養した(表4)。培養後、試料を洗浄し、西洋ワサビペルオキシダーゼ(HRP)が結合された2次抗体とともに培養した。検出のために、EnVisionTM+システム(Dako、Carpinteria、CA)を用いた。3,3−ジアミノベンジシン(DAB)またはRomulin AEC Chromogen(Biocare Medical、Concord、CA)を用いて信号を視覚化した。また、その試料をIP FLXヘマトキシリンで染色して背景を提供した。すべての顕微鏡写真はOlympus DP70高解像度デジタル顕微鏡カメラ(Olympus、Center Valley、PA)を具備したZeiss Axiophot顕微鏡(Carl Zeiss、Oberkochen、Germany)を用いて取得した。前記Borg、Sniper、Romulin Red、およびIP FLXヘマトキシリンはいずれもBiocare(Concord、CA)から購入した。
テラトーマの源(source)を確認するために、ヒト特異的ミトコンドリアマイトフュージン1遺伝子(MFN1)を、PCRを用いて増幅した。DNeasy血液および組織キット(Qiagen、USA)を用いて、ヒトiPSC、テラトーマ、およびテラトーマを有するRAG2突然変異ブタの尾由来の血液からゲノムDNAを分離した。PCR増幅のための条件は、96℃で2分間初期変性後、95℃で30秒変性、52℃で30秒アニーリング、および72℃で30分拡張の、32回の繰り返し(cycle)であった。PCR生成物を2.5%アガロースゲルにローディングした。そのPCR生成物の予測された大きさは236bpであった。分析のためのプライマーはF:GCTGGCTAAGAAGGCGATTA(配列番号2)およびR:TCCCCTTCTGGAGGTTAGAAA(配列番号3)であった。
受託番号:KCTC12496
受託日付:20131002
Claims (5)
- タレン(TALEN;Transcription Activator−Like Effector Nuclease)をブタ(Sus scrofa)の2番染色体の配列番号1のタレン認知配列部位に処理して二重対立遺伝子変形を誘導し、前記二重対立遺伝子変形が誘導された細胞を体細胞核移植(SCNT)に用いて突然変異体胚芽を生成する段階を含む、組換え活性遺伝子(RAG)2二重対立遺伝子変形を有する免疫細胞欠乏形質転換ミニクローンブタの生産方法。
- 前記タレンは、TALエフェクター−DNA変形酵素(Nuclease)をコーディングするベクターの形態で用いて当該細胞を形質感染させて行われることを特徴とする請求項1に記載の方法。
- 請求項1に記載の生産方法によって生産された組換え活性遺伝子(RAG)2二重対立遺伝子変形を有する免疫細胞欠乏形質転換ミニクローンブタ。
- タレン(TALEN;Transcription Activator−Like Effector Nuclease)をブタ(Sus scrofa)の2番染色体の配列番号1のタレン認知配列部位に処理して二重対立遺伝子変形を誘導し、前記二重対立遺伝子変形が誘導された細胞。
- 前記細胞は、RAG2ノックアウトミニブタ線維芽細胞の細胞株KCTC12496であることを特徴とする請求項4に記載の細胞。
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EP3068217A1 (en) | 2016-09-21 |
ES2935401T3 (es) | 2023-03-06 |
KR20150055665A (ko) | 2015-05-22 |
KR101554166B1 (ko) | 2015-09-22 |
EP3068217A4 (en) | 2017-04-26 |
US20160366860A1 (en) | 2016-12-22 |
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