JP5817955B2 - Production of hemophilia A model pig - Google Patents
Production of hemophilia A model pig Download PDFInfo
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- JP5817955B2 JP5817955B2 JP2010102569A JP2010102569A JP5817955B2 JP 5817955 B2 JP5817955 B2 JP 5817955B2 JP 2010102569 A JP2010102569 A JP 2010102569A JP 2010102569 A JP2010102569 A JP 2010102569A JP 5817955 B2 JP5817955 B2 JP 5817955B2
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- factor viii
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- hemophilia
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Description
本発明は、Ｘ染色体上の第VIII因子遺伝子をノックアウトした血友病Ａモデルブタ、特に自発性の出血症状（自然出血症状）を呈する血友病Ａモデルブタに関する。 The present invention relates to hemophilia A model pigs in which the factor VIII gene on the X chromosome has been knocked out, and particularly to hemophilia A model pigs exhibiting spontaneous bleeding symptoms (spontaneous bleeding symptoms).
血友病は血液凝固因子欠乏症の１つで、難治性の先天性出血性疾患である。Ｘ染色体に座する第VIII因子遺伝子異常による血友病Ａと第IX因子遺伝子異常による血友病Ｂに分けられる。１９世紀中頃まで重症血友病男児は成人する前に死亡していたが、診断の進歩や有効な治療法の進歩により患者の生活は向上した。特に血液製剤の開発は血友病治療の転換点であり現在の治療の基礎となった。血液製剤の出現はまさに画期的なことであったが、一方で、多くの血友病患者が血液製剤輸注により肝炎ウイルスやＨＩＶに感染し、患者は大きな犠牲を強いられることになった。その後、ウイルス不活化技術の向上と遺伝子組み換え凝固因子製剤の開発によりウイルス感染は激減し、既知のウイルスやプリオンへの暴露などの危険はほぼ無くなったと考えられる。現在は出血時に凝固因子製剤を投与するオンデマンド療法が中心であり、不測の致死的出血や傷害性出血を防ぐことは出来ない。また、補充する凝固因子の半減期が短いため（第VIII因子は８〜１２時間、第IX因子は１８〜２４時間）、重症患者では凝固因子製剤の輸注回数が多くなることで、補充した凝固因子に対する同種抗体（インヒビター）が発生することがあり、治療が極めて困難になる。この他、製剤療法には、凝固因子製剤のコストの問題、製剤への未知の病原物質の混入の問題なども残っている。こうした問題を解決し、患者のＱＯＬ（Quality of Life）を向上させ治癒へとつながる次世代の治療法として遺伝子治療が期待されている。 Hemophilia is a blood coagulation factor deficiency and is an intractable congenital hemorrhagic disease. It is divided into hemophilia A caused by abnormality in the factor VIII gene located on the X chromosome and hemophilia B caused by abnormality in the factor IX gene. Until the middle of the 19th century, severe hemophilia boys died before they became adults, but advances in diagnosis and advances in effective treatment have improved the lives of patients. In particular, the development of blood products was a turning point in the treatment of hemophilia and became the basis of current treatment. The advent of blood products was truly groundbreaking, but on the other hand, many hemophilia patients were infected with hepatitis virus and HIV by blood product infusion, and patients were forced to sacrifice greatly. Later, viral infections were drastically reduced by the improvement of virus inactivation technology and the development of genetically modified coagulation factor preparations, and the risk of exposure to known viruses and prions was almost eliminated. At present, on-demand therapy is mainly used to administer a clotting factor preparation at the time of bleeding, and it is not possible to prevent unexpected lethal or injured bleeding. Moreover, since the coagulation factor to be supplemented has a short half-life (Factor VIII is 8 to 12 hours, Factor IX is 18 to 24 hours), the number of infusions of the coagulation factor preparation is increased in critically ill patients. Alloantibodies against the factor (inhibitors) may occur, making treatment extremely difficult. In addition, the problem of the cost of the coagulation factor preparation and the problem of contamination of unknown pathogens into the preparation remain in the pharmaceutical therapy. Gene therapy is expected as a next-generation therapy that solves these problems, improves the quality of life (QOL) of patients, and leads to healing.
遺伝子治療の現在の技術レベルにおいて、治療レベルの遺伝子発現を得るためには、遺伝子導入にウイルスベクターを利用することが現実的である。ウイルスベクターの安全性については様々な改良がなされ、実用レベルに達している。しかし、各ベクターには搭載可能遺伝子サイズに制限があり、第VIII因子は遺伝子サイズが大きすぎる為、血友病Ａ遺伝子治療研究は遅れていた。生体内で活性化される際に分解除去される部分の遺伝子を除く等の改変第VIII因子が考案され、海外では臨床治験も行われるようになったが、いずれのトライアルも一年以上に及ぶ長期間の第VIII因子活性の上昇は見られず、造血障害や肝障害が出現した症例もあった。血友病Ａに対する遺伝子治療の臨床応用には、これらの課題をクリアすることが必須であり、更なる検討が必要であると考えられる。これまでの臨床治験は、第VIII因子遺伝子ノックアウトマウス（非特許文献１及び２参照）や自然発症の血友病イヌ（非特許文献３参照）を用いた実験結果を基に実施されている。 In order to obtain a therapeutic level of gene expression at the current level of gene therapy, it is practical to use viral vectors for gene transfer. Various improvements have been made to the safety of viral vectors, and they have reached a practical level. However, each vector has a restriction on the size of the gene that can be loaded, and the factor VIII gene size is too large, so research on hemophilia A gene therapy has been delayed. Modified factor VIII has been devised, such as removing genes that are decomposed and removed when activated in vivo, and clinical trials have been conducted overseas. There was no long-term increase in factor VIII activity, and there were cases in which hematopoietic disorder or hepatic disorder appeared. Clearing these issues is essential for the clinical application of gene therapy for hemophilia A, and further studies are considered necessary. Clinical trials so far have been carried out based on experimental results using factor VIII gene knockout mice (see Non-patent Documents 1 and 2) and spontaneous hemophilia dogs (see Non-Patent Document 3).
他方、本発明者らは体細胞核直接導入法によるクローンブタの作出について報告（非特許文献４参照）し、また、ＥｎｄｏＧａｌＣ遺伝子及びｈＤＡＦ遺伝子からなる導入遺伝子を共発現する、超急性拒絶反応及び急性拒絶反応が抑制されたブタ臓器を提供しうるダブルトランスジェニックブタ（特許文献１参照）を報告している。 On the other hand, the present inventors have reported the production of a cloned pig by the somatic cell nucleus direct introduction method (see Non-Patent Document 4), and co-expressed a transgene comprising an EndoGalC gene and an hDAF gene. A double transgenic pig capable of providing a porcine organ with suppressed rejection (see Patent Document 1) has been reported.
凝固第VIII因子（ＦVIII）は種特異性が強いことから、よりヒトに近い実験動物を用いた前臨床試験で治療効果と安全性が評価された後にヒト臨床治験が行われるべきである。非ヒト霊長類であるサルには血友病が同定されておらず、導入したＢＤＤＦVIII抗原量の測定は可能になったが、ＦVIII活性の測定は不可能である。ヒト臨床試験開始前にはＢＤＤＦVIIIの止血能・治療効果の評価は必須であり、そのためにはヒトの類縁動物の欠損モデルが必要となる。しかし、霊長類で血友病モデルを作製するには、技術的にも倫理的にも大きな問題がある。 Since coagulation factor VIII (FVIII) is highly species-specific, human clinical trials should be conducted after therapeutic efficacy and safety have been evaluated in preclinical studies using experimental animals that are closer to humans. Hemophilia has not been identified in monkeys that are non-human primates, and the amount of BDDFVIII antigen introduced can be measured, but FVIII activity cannot be measured. Before the start of human clinical trials, it is essential to evaluate the hemostatic ability / therapeutic effect of BDDFVIII. To that end, a deficient model of human related animals is required. However, creating a hemophilia model in primates has major technical and ethical problems.
本発明の課題は、血友病Ａの病態や効果的な治療方法を開発する上で有用な、自然出血症状を呈する血友病Ａモデルブタを提供することにある。 An object of the present invention is to provide a hemophilia A model pig exhibiting spontaneous bleeding symptoms, which is useful in developing the pathology of hemophilia A and an effective treatment method.
本発明者らは、血友病Ａの根治に向けた治療研究を進め、臨床への展開を加速させるために、ヒトに近い病態モデルとして近年本発明者らのグループで開発されたクローンブタ作製技術を用い、ＦVIII遺伝子を破壊した血友病Ａブタの作製を検討した。種々の工夫をしてＦVIIIノックアウトブタを作出した。その結果、自発的な出血症状の発症頻度が低いＦVIII遺伝子欠損マウスに比べて、自発的な出血症状の発症頻度が極めて高いＦVIII遺伝子欠損ブタが得られることを見いだし、本発明を完成した。これら血友病モデルブタは、出産後１日以内にＦVIIIを投与し、その後２〜３日間隔でＦVIIIを投与する必要があり、その措置を行わなければ、自然出血が多く観察されるため、死亡したり重篤な症状が観察される。また、飼養管理する柵内の床に通常とは異なる柔らかい素材のゴムマットを敷くなどの措置も講じる必要があることがわかった。 In order to promote therapeutic research for the cure of hemophilia A and accelerate clinical development, the present inventors have recently produced a cloned pig developed by the present inventors group as a pathological model close to humans. Using technology, the production of hemophilia A pigs with the FVIII gene disrupted was examined. FVIII knockout pigs were created with various measures. As a result, it was found that FVIII gene-deficient pigs with a very high frequency of spontaneous bleeding symptoms were obtained compared to FVIII gene-deficient mice with a low frequency of spontaneous bleeding symptoms, and the present invention was completed. These hemophilia model pigs need to administer FVIII within one day after delivery, and then administer FVIII every two to three days. Otherwise, spontaneous bleeding is often observed. Death or serious symptoms are observed. It was also found that it was necessary to take measures such as placing a rubber mat made of a soft material different from the usual on the floor inside the fence to be kept.
すなわち、本発明は（１）Ｘ染色体上の第VIII因子遺伝子の一部もしくは全部が欠損し、野生型において発現される第VIII因子を発現する機能が失われたことを特徴とする、自発的な出血症状を呈する血友病Ａモデルブタや、（２）上記（１）記載の自発的な出血症状を呈する血友病Ａモデルブタに由来する臓器、組織又は細胞に関する。
That is, the present invention is (1) spontaneous, characterized in that part or all of the factor VIII gene on the X chromosome is deleted and the function of expressing factor VIII expressed in the wild type is lost. The present invention relates to an organ, tissue or cell derived from a hemophilia A model pig exhibiting various bleeding symptoms and (2) a hemophilia A model pig exhibiting spontaneous bleeding symptoms described in (1) above.
また、本発明は、（３）以下の工程を備えた、自発的な出血症状を呈する血友病Ａモデルブタの作出方法：
（ａ）第VIII因子遺伝子のエキソン１６を標的としたターゲティングベクターを作製する工程；
（ｂ）胎児線維芽細胞に、工程（ａ）で作製したターゲティングベクターを導入し、相同組換えにより生じた遺伝子組換え細胞を選抜する工程；
（ｃ）採取した豚の体内成熟卵子から除核する工程；
（ｄ）工程（ｃ）で除核された卵子に、工程（ｃ）で選抜された遺伝子組換え細胞の細胞核を注入する工程；
（ｅ）工程（ｄ）で細胞核が注入された卵子に活性化処理を施し、活性化処理後の核移植胚を雌豚の卵管又は子宮に移植する工程；
や、（４）Ｘ染色体上の第VIII因子遺伝子の一部もしくは全部が欠損し、野生型において発現される第VIII因子を発現する機能が失われた、自発的な出血症状を呈する第VIII因子ノックアウトブタに被検物質を投与し、該モデルブタにおける血液凝固機能の程度を評価することを特徴とする血友病の予防・治療剤のスクリーニング方法に関する。
The present invention also provides (3) a method for producing a hemophilia A model pig exhibiting spontaneous bleeding symptoms, comprising the following steps:
(A) producing a targeting vector targeting exon 16 of the factor VIII gene;
(B) introducing the targeting vector prepared in step (a) into fetal fibroblasts and selecting genetically modified cells produced by homologous recombination;
(C) a step of enucleating from the collected porcine mature eggs;
(D) A step of injecting the nuclei of the genetically modified cells selected in the step (c) into the egg enucleated in the step (c);
(E) a step of activating the egg into which the cell nucleus has been injected in step (d), and transplanting the nuclear transfer embryo after the activation treatment to the oviduct or uterus of a sow;
And (4) a factor VIII exhibiting spontaneous bleeding symptoms in which part or all of the factor VIII gene on the X chromosome is deleted and the function of expressing the factor VIII expressed in the wild type is lost. The present invention relates to a screening method for a prophylactic / therapeutic agent for hemophilia, comprising administering a test substance to a knockout pig and evaluating the degree of blood coagulation function in the model pig.
さらに、本発明は（５）ヒト第VIII因子により、Ｘ染色体上の第VIII因子遺伝子の一部もしくは全部が欠損し、野生型において発現される第VIII因子を発現する機能が失われた、自発的な出血症状を呈する第VIII因子ノックアウトブタを免疫し、ヒト第VIII因子とブタ第VIII因子とに交差反応性を有する抗第VIII因子抗体を調製することを特徴とする抗第VIII因子ブタ抗体の製造方法に関する。 Furthermore, the present invention is (5) human factor VIII, part or all of the factor VIII gene on the X chromosome deficient, the ability to express the Factor VIII that is expressed in the wild type was lost, An anti-factor VIII pig characterized by immunizing a factor VIII knockout pig with spontaneous bleeding symptoms and preparing an anti-factor VIII antibody having cross-reactivity with human factor VIII and porcine factor VIII The present invention relates to a method for producing an antibody.
ヒトに近い病態モデルが作製されれば、遺伝子治療研究だけでなく、新規治療法や新規製剤開発、インヒビター治療研究に絶大な貢献をもたらし、血友病Ａ患者の望む治癒へ向けた治療研究が加速すると期待される。本発明によると、自然出血症状を呈する血友病Ａモデルブタを提供することができる。また、本発明の血友病Ａモデルブタに被検物質を投与し、該モデルブタにおける血液凝固機能の程度を比較・評価することにより、血友病の予防・治療剤をスクリーニングすることもできる。 If a pathological model close to humans is created, not only gene therapy research, but also new therapeutic methods, new drug development, and inhibitor therapy research will be greatly contributed, and therapeutic research aimed at the healing desired by hemophilia A patients Expected to accelerate. According to the present invention, a hemophilia A model pig exhibiting spontaneous bleeding symptoms can be provided. Further, a prophylactic / therapeutic agent for hemophilia can be screened by administering a test substance to the hemophilia A model pig of the present invention and comparing and evaluating the degree of blood coagulation function in the model pig. .
本発明の血友病Ａモデルブタとしては、Ｘ染色体上のＦVIII遺伝子の一部もしくは全部が欠損し、野生型において発現されるＦVIIIを発現する機能が失われたブタであれば特に制限されず、より具体的には雄ヘミ接合体ブタ（ＦVIII：Ｘ−／Ｙ）や雌ホモ接合体（ＦVIII：Ｘ−／Ｘ−）を例示することができるが、これらの中でも自発性の出血症状を呈する血友病モデルブタを好適に例示することができる。上記雌ホモ接合体は、雌ヘテロ接合体ブタ（ＦVIII：Ｘ−／Ｘ＋）と雄ヘミ接合体ブタ（ＦVIII：Ｘ−／Ｙ）を掛け合わせると、２５％の産生比率で出生する。 The hemophilia A model pig of the present invention is not particularly limited as long as it is a pig that lacks part or all of the FVIII gene on the X chromosome and loses the function of expressing FVIII expressed in the wild type. , more specifically male hemizygous porcine (FVIII: X - / Y) and female homozygous (FVIII: X - / X - ) can be exemplified, bleeding symptoms spontaneous among these The hemophilia model pig to present can be illustrated suitably. The female homozygote is born at a production ratio of 25% when a female heterozygous pig (FVIII: X − / X + ) is multiplied by a male hemizygous pig (FVIII: X − / Y).
上記血友病モデルブタは、（ａ）ＦVIII遺伝子のエキソン１６を標的としたターゲティングベクターを作製する工程；（ｂ）胎児線維芽細胞に、工程（ａ）で作製したターゲティングベクターを導入し、相同組換えにより生じた遺伝子組換え細胞を選抜する工程；（ｃ）採取した豚の体内成熟卵子から除核する工程；（ｄ）工程（ｃ）で除核された卵子に、工程（ｃ）で選抜された遺伝子組換え細胞の細胞核を注入する工程；（ｅ）工程（ｄ）で細胞核が注入された卵子に活性化処理を施し、活性化処理後の核移植胚を雌豚の卵管又は子宮に移植する工程；の各工程を備えたＦVIIIノックアウトブタの作出方法により得ることができる。 In the hemophilia model pig, (a) a step of producing a targeting vector targeting exon 16 of the FVIII gene; (b) introducing the targeting vector produced in step (a) into fetal fibroblasts, Selecting a genetically modified cell produced by recombination; (c) enucleating from the collected porcine in-vivo mature egg; (d) applying the enucleated egg in step (c) to step (c); A step of injecting the cell nucleus of the selected genetically modified cell; (e) an activation treatment is performed on the egg into which the cell nucleus has been injected in step (d), and the nuclear transfer embryo after the activation treatment is used as a fallen oviduct of a sow It can be obtained by a method for producing an FVIII knockout pig comprising the steps of transplanting to the uterus.
上記工程（ａ）における血友病モデルブタの作出に用いられるターゲティングベクターとしては、相同組換えによりＦVIII遺伝子を破壊し、ＦVIII遺伝子機能を欠失させうるもので、ＦVIIIをコードする遺伝子中のエキソン１６を含む領域が改変されたポリヌクレオチドを含有し、かつ該ポリヌクレオチドが、ＦVIIIの生理活性を欠損した産物を生じるものを挙げることができ、相同組換えにより生じた遺伝子組換え細胞をスクリーニングしやすくするために、エキソン１６の中にネオマイシン耐性遺伝子（ＰＧＫ−Ｎｅｏ）等の抗生物質耐性遺伝子やβガラクトシダーゼ遺伝子等の陽性選択マーカーを挿入させることができる他、ＨＳＶチミジンキナーゼ（ＨＳＶ−ＴＫ）遺伝子、ジフテリア毒素（ＤＴ−Ａ）遺伝子等の陰性選択マーカーを有するベクターが好ましい。 The targeting vector used in the production of the hemophilia model pig in the above step (a) is one that can disrupt the FVIII gene by homologous recombination and delete the FVIII gene function. An exon in the gene encoding FVIII A region containing 16 contains a modified polynucleotide, and the polynucleotide can produce a product that lacks the physiological activity of FVIII, and screens for genetically modified cells produced by homologous recombination. In order to facilitate, exon 16 can be inserted with an antibiotic resistance gene such as neomycin resistance gene (PGK-Neo) and a positive selection marker such as β-galactosidase gene, and HSV thymidine kinase (HSV-TK) gene. And negative selection markers such as diphtheria toxin (DT-A) gene Vectors are preferred.
上記工程（ｂ）において、胎児線維芽細胞におけるターゲティングベクターによるＦVIIIをコードする遺伝子の相同組換えは、ターゲティングベクターをブタ胎児線維芽細胞に導入することにより達成される。ターゲティングベクターを胎児線維芽細胞に導入する方法は、特に限定されるものではなく、例えば、エレクトロポレーション法、リポソーム法、リン酸カルシウム法、ＤＥＡＥ−デキストラン法等を挙げることができる。ターゲティングベクター導入後の胎児線維芽細胞を、細胞内での相同組換えが起こっているかを確認するためのサザンブロッティング法やＰＣＲ法等の常法によりスクリーニングすることにより、ＦVIIIをコードする遺伝子が欠損した胎児線維芽細胞（遺伝子組換え細胞）が得られる。上記胎児線維芽細胞はトリプシン処理で細胞を分散させたものが好ましく、また、培養細胞が線維芽細胞であることを、サイトケラチンとＳＳＥＡ−１との陰性反応、ビメンチンでの陽性反応、線維芽細胞の特異的プライマーによるＰＣＲ分析等により確認することが好ましい。また、レシピエントや仮親と毛色の異なる品種のブタの線維芽細胞を核移植用ドナーとすると、毛色からノックアウトブタであるかどうかを簡便に判定することができる。 In the above step (b), homologous recombination of the gene encoding FVIII with a targeting vector in fetal fibroblasts is achieved by introducing the targeting vector into porcine fetal fibroblasts. The method for introducing the targeting vector into fetal fibroblasts is not particularly limited, and examples thereof include an electroporation method, a liposome method, a calcium phosphate method, and a DEAE-dextran method. By screening the fetal fibroblasts after introduction of the targeting vector by conventional methods such as Southern blotting or PCR to confirm whether homologous recombination has occurred in the cells, the gene encoding FVIII is deleted. Fetal fibroblasts (genetical recombination cells) are obtained. The fetal fibroblasts are preferably those in which the cells are dispersed by trypsin treatment, and that the cultured cells are fibroblasts, a negative reaction between cytokeratin and SSEA-1, a positive reaction with vimentin, a fibroblast It is preferable to confirm by PCR analysis using a specific primer of the cell. Moreover, if a fibroblast of a pig having a hair color different from that of the recipient or temporary parent is used as a donor for nuclear transplantation, it can be easily determined from the hair color whether it is a knockout pig.
また、核移植に用いるドナー線維芽細胞の細胞周期は特に制限されるものではないが、細胞周期Ｇ0／Ｇ01期に同調させた細胞が好ましい。細胞周期Ｇ0／Ｇ01期に同調させた細胞は、例えばコンフルエントな状態で培養液の交換なしに、線維芽細胞を１６日間前後培養し続けることによって得ることができる。 The cell cycle of donor fibroblasts used for nuclear transfer is not particularly limited, but cells synchronized with the cell cycle G 0 / G 01 are preferred. Cells synchronized to the cell cycle G 0 / G 01 phase can be obtained, for example, by continuously culturing fibroblasts for about 16 days in a confluent state without changing the culture medium.
上記工程（ｃ）におけるブタの卵子（レシピエント卵子）としては、ブタの成熟卵子であれば特に制限されるものではなく、ＰＧＦ２α、ＰＧＦ２α類縁体クロプロステロール、ｅＣＧ等のホルモン投与による過排卵処理により得られる体内成熟卵子の他、屠場由来の卵巣から採取した卵子を体外成熟させたものも使用できるが、着床率の点からして体内成熟卵子、特に性成熟した雌ブタ、例えば生後１６０〜１９４日齢の春期発動前の雌ブタから採取した体内成熟卵子が好ましい。かかる体内成熟卵子は、過排卵処理により得られる雌ブタの子宮及び卵巣を、ＰＢＳ溶液等を用いて卵管灌流を行うことにより採取することができるが、卵丘細胞が付着している卵子はヒアルロニダーゼ処理を行って、卵丘細胞を除去することが好ましい。 The porcine ovum (recipient ovum) in the step (c) is not particularly limited as long as it is a porcine mature ovum. Superovulation treatment by administration of hormones such as PGF2α, PGF2α analog cloprosterol, eCG and the like. In addition to the in-vivo matured ovum obtained by the above method, an in-vitro matured egg collected from an ovary derived from a slaughterhouse can be used. However, in terms of implantation rate, an in-vivo matured egg, particularly a sexually matured sow, for example, 160 Preferred are in-vivo mature eggs collected from sows prior to spring activation at 194 days of age. Such in-vivo mature eggs can be collected from the uterus and ovaries of sows obtained by superovulation treatment by performing oviduct perfusion using a PBS solution or the like. Hyaluronidase treatment is preferably performed to remove cumulus cells.
上記ブタのレシピエント卵子からの除核は、細胞骨格形成阻害剤であるサイトカラシンＢ処理を施したブタの卵子から除核することが好ましく、より具体的にはサイトカラシンＢを含有するＰＺＭ３液、ＮＣＳＵ２３等の培地で体外成熟卵子等のレシピエント卵子を処理した後、除核操作用シャーレのサイトカラシン入りドロップに移してホールディングピペットで保定し、透明帯を迅速・的確に貫通することができる除核用ピペット（外径２５〜３０μｍ）を用いて、Ｍ（metaphase）II期の染色体を含む第一極体の付近を極体ごと吸引することにより行われる。なお、吸引した極体を調べることにより除核できていることを確認することが好ましく、また除核卵子からはサイトカラシンＢを除去することが好ましい。 The enucleation from the pig recipient egg is preferably carried out from a pig egg treated with cytochalasin B, which is a cytoskeleton formation inhibitor, more specifically, a PZM3 solution containing cytochalasin B. After treatment of recipient eggs such as in vitro matured eggs in a medium such as NCSU23, transfer to a drop containing cytochalasin in a petri dish for enucleation operation and hold it with a holding pipette, and can penetrate the zona pellucida quickly and accurately Using a pipette for enucleation (outer diameter 25-30 μm), the vicinity of the first polar body containing the chromosome of M (metaphase) II stage is sucked together with the polar body. It is preferable to confirm that enucleation has been achieved by examining the aspirated polar body, and it is preferable to remove cytochalasin B from the enucleated ovum.
上記工程（ｄ）において、除核された卵子にブタ胎児遺伝子組換え線維芽細胞核を注入する方法としては、ブタ胎児遺伝子組換え線維芽細胞の核を除核されたレシピエント卵母細胞に直接注入（インジェクション）する方法を特に好適に例示することができる。かかる核の直接注入には、例えば、プライムテック株式会社製のＰＭＭ三次元マイクロマニピュレーションシステムを用いることができる。特に、インジェクションピペットとしては、透明帯の貫通が迅速・精確かつ簡単にでき、細胞質膜へのダメージを最小にすることができるものが好ましく、かかるインジェクションピペットとしてはピエゾマイクロマニピュレーター（プライムテック株式会社製）に取り付けた体細胞注入用ピペット（外径２５〜３０μｍ、先端を３０〜４５度の角度で研磨）を具体的に例示することができる。 In the step (d), the method of injecting the fetal bovine genetically modified fibroblast nucleus into the enucleated egg is directly applied to the enucleated recipient oocyte. A method of injection (injection) can be particularly preferably exemplified. For such direct injection of nuclei, for example, a PMM three-dimensional micromanipulation system manufactured by Primetec Corporation can be used. In particular, the injection pipette is preferably one that can quickly and accurately penetrate the zona pellucida and minimize damage to the cytoplasmic membrane. As such an injection pipette, a piezoelectric micromanipulator (manufactured by Primetec Corporation) is preferable. A pipette for injecting somatic cells (outer diameter: 25 to 30 μm, the tip polished at an angle of 30 to 45 degrees) attached to () can be specifically exemplified.
かかる工程（ｄ）において体細胞核が注入された卵子には、上記工程（ｅ）において活性化処理を施すことが好ましい。かかる活性化処理としては、従来公知の核移植胚の活性化処理方法であれば特に制限されるものではないが、電気パルス活性化処理を好適に例示することができる。電気パルス活性化処理としては、電荷の大きい１回の電気パルス、例えば１．５ｋＶ／ｃｍ、１００μｓｅｃ、１回を印加する方がそれより小さい電荷の電気パルスを２回印加するよりも胚活性の点で好ましく、また、電気パルス活性化処理における培地としてはＮＣＳＵ２３（J.Reprod.Fertil.Suppl.,48,61,1993）を用いることが高い胚盤胞形成率の点で好ましい。また、電気パルスによる活性化処理の場合、体内成熟卵子の方が体外成熟卵子に比べて胚盤胞の発生能の点で好ましい。さらに、レシピエント細胞として体内成熟卵子を用いる場合には、過排卵処理のために使用した最初のｈＣＧ投与後、５０〜６０時間後、好ましくは５４〜５５時間後に活性化処理をすることが望ましい。 It is preferable that the ovum into which the somatic cell nucleus is injected in the step (d) is subjected to an activation treatment in the step (e). Such activation treatment is not particularly limited as long as it is a conventionally known activation treatment method for a nuclear transfer embryo, and an electric pulse activation treatment can be preferably exemplified. In the electric pulse activation process, one electric pulse having a large charge, for example, 1.5 kV / cm, 100 μsec, one application of embryo activity is more effective than two electric pulses having a smaller electric charge. Further, NCSU23 (J. Reprod. Fertil. Suppl., 48, 61, 1993) is preferably used as a medium in the electric pulse activation treatment in terms of a high blastocyst formation rate. In addition, in the case of activation treatment by electric pulses, in-vivo matured eggs are preferred in terms of the ability to develop blastocysts compared to in vitro matured eggs. Further, when using a mature oocyte as a recipient cell, it is desirable to carry out an activation treatment 50 to 60 hours, preferably 54 to 55 hours after the first hCG administration used for superovulation treatment. .
また、卵細胞からの除核時及び該除核細胞への体細胞核の直接注入時の２回にわたって損傷を受けた核移植胚の胚盤胞への発生率を高め、クローンブタを効率よく作出するために、活性化処理後の核移植胚は、卵管又は子宮への移植前に、包埋剤により包埋処理を施すこともできる。かかる包埋剤による包埋処理は、複数回の包埋処理を行い、複数被膜で包埋した核移植胚とすることが好ましく、特に３重包埋処理を行い、３重被膜核移植胚とすることが好ましい。また、かかる複数回の包埋処理を行うに際しては、包埋剤の濃度を順次高めていく包埋処理、すなわち外層膜ほど高濃度の包埋剤を用いて包埋し、その物理的強度を内層から外層へと順次高めていくことが好ましい。 In addition, the incidence of blastocysts of nuclear transplanted embryos damaged twice during the enucleation from egg cells and the direct injection of somatic cell nuclei into the enucleated cells is increased, and a cloned pig is efficiently produced. Therefore, the nuclear transfer embryo after the activation treatment can be embedded with an embedding agent before transfer to the oviduct or uterus. The embedding process with such an embedding agent is preferably a nuclear transfer embryo that has been embedded multiple times and embedded in a plurality of coatings. It is preferable to do. In addition, when performing such multiple embedding processes, the embedding process in which the concentration of the embedding agent is sequentially increased, that is, the outer layer film is embedded using a higher concentration embedding agent, and the physical strength is increased. It is preferable to gradually increase from the inner layer to the outer layer.
包埋処理に用いられる包埋剤としては、核移植胚を包埋することにより、２回にわたって損傷を受けた核移植胚の桑実胚や胚盤胞への発生率を高め、クローンブタを効率よく作出することができるものであれば特に制限されるものではないが、損傷を受けた透明帯の修復・保護機能を有するものや、包埋処理後の核移植胚を卵管及び子宮に移植した後、生体内での分解機能を有するものや、卵管及び子宮の膜運動による損耗からの保護機能を有するものや、白血球の攻撃からの防御機能を有するものや、包埋皮膜を通しての栄養分の透過・排泄物の排出機能を有するものの他、包埋処理時に核移植胚に影響を与えることなく包埋処理することができるものや、顕微鏡下で包埋皮膜が確認しやすいものや、顕微鏡下で包埋胚の操作がし易くなるものなどが好ましい。かかる包埋剤としては、アルギン酸、寒天等の天然多糖類の他、ムコタンパク質、ポリアミノ酸などの蛋白質、生分解性有機高分子等を例示することができるが、上記包埋剤としての好ましい機能を備えたアルギン酸が特に好ましい。その他、包埋剤の使用濃度についても特に制限されるものではないが、上記包埋剤としての好ましい機能を十分発揮しうる濃度が好ましい。なお、複数回の包埋処理を行う場合、例えばアルギン酸と寒天等、包埋剤の種類を変えて包埋処理をすることもできる。 As an embedding agent used for embedding treatment, by embedding a nuclear transfer embryo, the incidence of twice-damaged nuclear transfer embryos to morulas and blastocysts is increased. Although it is not particularly limited as long as it can be efficiently produced, those that have the function of repairing / protecting damaged zona pellucida, and those that have been embedding treated in the oviduct and uterus After transplantation, those that have a degrading function in vivo, those that have a protective function from wear due to membrane movement of the fallopian tube and uterus, those that have a protective function from leukocyte attack, In addition to those that have a nutrient permeation and excretion excretion function, those that can be embedded without affecting the nuclear transfer embryo during the embedding process, those that are easy to check the embedding film under a microscope, Easier to manipulate embedded embryos under a microscope Such as is preferred. Examples of such embedding agents include natural polysaccharides such as alginic acid and agar, proteins such as mucoproteins and polyamino acids, biodegradable organic polymers, and the like. Preferred functions as the above embedding agents Alginic acid with is particularly preferred. In addition, the use concentration of the embedding agent is not particularly limited, but a concentration capable of sufficiently exhibiting a preferable function as the embedding agent is preferable. When performing the embedding process a plurality of times, the embedding process can be performed by changing the type of embedding agent such as alginic acid and agar.
例えば、アルギン酸を包埋剤として３重包埋処理する方法としては、アルギン酸ナトリウムを所定濃度（例えば０．５％、１．５％、２．０％）となるようにリンゲル液等に溶解し、あらかじめ滅菌しておき、この滅菌済みのアルギン酸ナトリウム溶液（例えば０．５％）に核移植胚を浸漬して十分馴染ませた後、塩化カルシウム液等のカルシウムイオン含有液と接触させ、アルギン酸ゲル包埋胚とした後、この包埋胚を前記アルギン酸ナトリウム溶液よりも高濃度のアルギン酸ナトリウム溶液（例えば１．５％）に浸漬して十分馴染ませた後、塩化カルシウム液等のカルシウムイオン含有液と接触させ、アルギン酸ゲル２重包埋胚とし、次いでこの２重包埋胚を上記アルギン酸ナトリウム溶液よりも高濃度のアルギン酸ナトリウム溶液（例えば２．０％）に浸漬して十分馴染ませた後、塩化カルシウム液等のカルシウムイオン含有液と接触させ、アルギン酸ゲル３重包埋核移植胚とする方法を具体的に示すことができる。 For example, as a method of triple embedding treatment using alginic acid as an embedding agent, sodium alginate is dissolved in Ringer's solution or the like so as to have a predetermined concentration (for example, 0.5%, 1.5%, 2.0%), After sterilizing in advance and immersing the nuclear transfer embryo in this sterilized sodium alginate solution (for example, 0.5%) and making it fully acclimatize, it is brought into contact with a calcium ion-containing solution such as calcium chloride solution, and the alginate gel capsule After making the embedded embryo, the embedded embryo was immersed in a sodium alginate solution (for example, 1.5%) having a concentration higher than that of the sodium alginate solution and sufficiently adjusted, and then a calcium ion-containing solution such as a calcium chloride solution was used. Alginate gel double-embedded embryo, which is then contacted with a sodium alginate solution having a higher concentration than the sodium alginate solution (eg After 2.0%) dipped by sufficiently fit to be contacted with a calcium ion-containing solution such as calcium chloride solution, a method of the alginic acid gel triple hull Umakaku transfer embryos can be shown specifically.
活性化処理後に、必要に応じて包埋剤による包埋処理をした核移植胚を、卵管又は子宮に移植する雌ブタとしては特に制限されるものではないが、人工授精させた後の妊娠２１〜４０日目にプロスタグランジンＦ２α等を用いて人工流産させ、同期化を行った雌ブタを用いることが好ましい。また、核移植胚を雌ブタの卵管又は子宮に移植するに際し、複数個の受精卵を核移植胚に混合して雌ブタの卵管又は子宮に移植する追い移植法を用いることが好ましい。 After the activation treatment, there is no particular limitation as a sow transplanted to a fallopian tube or uterus of a nuclear transfer embryo that has been embedded with an embedding agent as necessary, but pregnancy after artificial insemination It is preferable to use female pigs that have been artificially aborted using prostaglandin F2α on days 21 to 40 and synchronized. Further, when transplanting a nuclear transfer embryo into the oviduct or uterus of a sow, it is preferable to use a follow-up transfer method in which a plurality of fertilized eggs are mixed with the nuclear transfer embryo and transplanted to the oviduct or uterus of a sow.
また産仔したブタが、ＦVIIIノックアウトブタであることの確認は、産仔の毛色の他、ＦVIIIノックアウトクローンブタ、ＦVIIIノックアウトクローンブタの仮親の耳から採取したＤＮＡ並びにＦVIIIノックアウトクローンブタを作出するために用いた線維芽細胞のＤＮＡを採取し、ＰＣＲを行い、ＦVIIIノックアウトクローンブタが線維芽細胞と同一の遺伝子をもち、ＦVIII遺伝子が破壊され、自然出血症状を呈するかどうかを確認することにより同定することができる。 In addition to confirming that the pups were FVIII knockout pigs, in addition to the color of the puppies, FVIII knockout clone pigs, FVIII knockout clone pigs, and DNA collected from the foster parent's ears and FVIII knockout clone pigs were created. DNA was collected from the fibroblasts used in the experiment, and PCR was performed to identify whether the FVIII knockout clone pig had the same gene as the fibroblasts, the FVIII gene was disrupted, and spontaneous bleeding was observed. can do.
核移植用ドナーとして雄の胎児線維芽細胞を用いたときには雄ヘミ接合体ブタ（ＦVIII：Ｘ−／Ｙ）からなる血友病モデルブタを作出することができるが、雌の胎児線維芽細胞を用いたときには血友病症状を呈さない雌ヘテロ接合体ブタ（ＦVIII：Ｘ−／Ｘ＋）が得られる。この場合、雄ヘミ接合体ブタと雌ヘテロ接合体ブタを掛け合わせると、血友病症状を呈する２５％の雌ホモ接合体、２５％の雌ヘテロ接合体、血友病症状を呈する２５％の雄ヘミ接合体ブタ、２５％の雄の正常ブタの出生割合で産仔が得られる。前記非特許文献２によると、雄ヘミ接合体マウスで約１１％、雌ホモ接合体マウスで約７％が自発性の出血症状を呈するに過ぎず、８〜１０週を経過したＦVIIIＫＯマウスでは、尾を切断したり、尾を火傷させた場合でも全マウスが生存すると報告されているが、本発明によると、雄ヘミ接合体ブタで１００％自発性の出血症状を呈し、５週を経過した後でも自然出血症状を維持していた。 Male hemizygotes pigs when using male fetal fibroblasts as donor for nuclear transfer: - but (FVIII X / Y) hemophilia model pigs made of can be created, and the female fetal fibroblast When used, female heterozygous pigs (FVIII: X − / X + ) that do not exhibit hemophilia symptoms are obtained. In this case, when male hemizygous pigs and female heterozygous pigs are crossed, 25% female homozygotes exhibiting hemophilia symptoms, 25% female heterozygotes, 25% exhibiting hemophilia symptoms Offspring are obtained at the birth rate of male hemizygous pigs, 25% male normal pigs. According to Non-Patent Document 2, only about 11% of male hemizygous mice and about 7% of female homozygous mice exhibit spontaneous bleeding symptoms, and in FVIIIKO mice that have passed 8-10 weeks, Although it has been reported that all mice survive even when the tail is cut or the tail is burned, according to the present invention, male hemizygous pigs exhibited 100% spontaneous bleeding symptoms and 5 weeks passed. He continued to have spontaneous bleeding.
本発明はまた、本発明の血友病モデルブタに由来する臓器、組織又は細胞に関し、かかる臓器、組織又は細胞は、血友病モデルブタと同様に、血友病の予防・治療剤のスクリーニングに有利に用いることができる。また、臓器、組織または生殖細胞を含む細胞を用いることで、後代を含む個体作出に利用できる。 The present invention also relates to an organ, tissue or cell derived from the hemophilia model pig of the present invention, and the organ, tissue or cell is screened for a prophylactic / therapeutic agent for hemophilia in the same manner as the hemophilia model pig. Can be advantageously used. In addition, by using cells containing organs, tissues or germ cells, it can be used for individual production including progeny.
本発明の血友病の予防・治療剤のスクリーニング方法としては、上記本発明の血友病モデルブタ（ＦVIIIノックアウトブタ）をに被検物質を投与し、該モデルブタにおける血液凝固機能の程度を、被検物質を投与しない場合と比較・評価する方法であれば特に制限されず、上記血液凝固機能の程度とは、自然出血や怪我等の外因性要因による出血の症状の止血の程度や止血の持続期間の程度をいい、血液凝固機能の程度を既存の血友病の予防・治療剤と比較・評価することができる。上記被検物質としては、ＦVIII遺伝子やその一部、又はＦVIII遺伝子改変体が組み込まれたＤＮＡベクター、例えば、組換えアデノ随伴ウイルス（adeno-associated virus：ＡＡＶ）ベクター、組換えレンチウイルス（lentivirus）ベクター、組換えアデノウイルス（adeno virus）ベクター、ネイキッドＤＮＡベクター等を好適に例示することができる。本発明は、上記本発明のスクリーニング方法により得られた血友病Ａの予防・治療剤をも包含する。 As a screening method for a prophylactic / therapeutic agent for hemophilia of the present invention, a test substance is administered to the hemophilia model pig (FVIII knockout pig) of the present invention, and the degree of blood coagulation function in the model pig is determined. The method of comparison / evaluation is not particularly limited as long as the test substance is not administered, and the degree of the blood coagulation function refers to the degree of hemostasis of hemorrhage due to exogenous factors such as spontaneous bleeding or injury, and hemostasis The degree of blood coagulation function can be compared and evaluated with existing preventive / therapeutic agents for hemophilia. Examples of the test substance include a DNA vector in which the FVIII gene, a part thereof, or a modified FVIII gene is incorporated, such as a recombinant adeno-associated virus (AAV) vector, a recombinant lentivirus (lentivirus) Preferred examples include vectors, recombinant adenovirus vectors, naked DNA vectors and the like. The present invention also includes a prophylactic / therapeutic agent for hemophilia A obtained by the screening method of the present invention.
本発明の抗ＦVIII抗体としては、ヒトＦVIIIとブタＦVIIIとに交差反応性を有する抗体であれば特に制限されず、抗ＦVIIIモノクローナル抗体が好ましい。本発明の抗ＦVIII抗体は、ヒトＦVIIIでＦVIIIノックアウトブタを免疫することにより調製することができる。かかる本発明のヒトＦVIIIとブタＦVIIIとに交差反応性を有する抗体は、ＦVIIIインヒビターである抗ＦVIII抗体を無力化あるいは回避する血友病の予防・治療剤のスクリーニングに使用することができる。 The anti-FVIII antibody of the present invention is not particularly limited as long as it has cross-reactivity with human FVIII and porcine FVIII, and an anti-FVIII monoclonal antibody is preferable. The anti-FVIII antibody of the present invention can be prepared by immunizing FVIII knockout pigs with human FVIII. Such an antibody having cross-reactivity with human FVIII and porcine FVIII of the present invention can be used for screening for a prophylactic / therapeutic agent for hemophilia that disables or avoids an anti-FVIII antibody that is an FVIII inhibitor.
［ＦVIII遺伝子ＫＯクローンブタの作出］
１．ＦVIII遺伝子ターゲティングベクターの作製（ベクター名：Ｆ８−ＧＴＶ）
胎齢６５日目の雄ブタ胎仔線維芽細胞からDNAzol（Invitrogen社製）を用いてブタゲノムＤＮＡを分離し、このゲノムＤＮＡからＦVIII遺伝子のエキソン１６を標的とするベクターを構築した。Sus scrofa coagulation factor VIII cDNA sequence（Ａｃｃｅｓｓｉｏｎ ｎｕｍｂｅｒ；ＮＭ＿２１４１６７）をもとにプライマーを作製し（表１）、エキソン１４からエキソン２１までと、エキソン１６からエキソン２２までをExpand Long Template PCR System（Roche Diagnostics社製）を用いたＰＣＲにて増幅させ、pCR2.1-TOPO Cloning Kit（Invitrogen社製）またはpCRBluntII-TOPO Cloning Kit（Invitrogen社製）を用いてクローニング後、ABI PRISM Genetic Analyzer（Applied Biosystems社製）によるＤＮＡ塩基配列決定と、エキソン部位とｃＤＮＡ配列とを照合した。さらにイントロン１４部位におけるXhoI認識配列を付加したプライマー（表１）とエキソン２１プライマーのＰＣＲによりクローニングしたイントロン１４−エキソン２１をエキソン１６内にあるXhoIで処理したイントロン１４−エキソン１６の５’−アームを、エキソン１６プライマーとイントロン２１部位におけるXbaI認識配列を付加したプライマー（表１）によりエキソン１６−イントロン２１の３’−アームをクローニングし、pBS-HSVTK-PGKNeo（京都大学ウイルス研究所 生田宏一教授からの供与）のXhoIとXbaI部位へ組み込み、エキソン１６の中にＰＧＫ−Ｎｅｏを挿入させ、５’−アーム上流に自殺遺伝子であるＨＳＶ−ＴＫを配したＦVIII遺伝子ターゲティングベクターを作製した。エレクトロポレーションに用いるため、NotI（TOYOBO社製）の消化により線状化し、フェノール／クロロフォルムによる精製後、ＨＢＳ（HEPEs Buffered Saline）で２５ｎＭの濃度に希釈した。
[Production of FVIII gene KO cloned pig]
1. Preparation of FVIII gene targeting vector (vector name: F8-GTV)
Porcine genomic DNA was isolated from male pig fetal fibroblasts at 65 days of gestation using DNAzol (Invitrogen), and a vector targeting exon 16 of the FVIII gene was constructed from this genomic DNA. Primers were prepared based on Sus scrofa coagulation factor VIII cDNA sequence (Accession number; NM — 214167) (Table 1). Expand Long Template PCR System (Roche Diagnostics) from exon 14 to exon 21 and from exon 16 to exon 22 ABI PRISM Genetic Analyzer (Applied Biosystems) after cloning using pCR2.1-TOPO Cloning Kit (Invitrogen) or pCRBluntII-TOPO Cloning Kit (Invitrogen) The DNA nucleotide sequence determined by the above method was compared with the exon site and the cDNA sequence. Furthermore, the 5'-arm of intron 14-exon 16 in which intron 14-exon 21 cloned by PCR of the primer (Table 1) to which XhoI recognition sequence at the intron 14 site was added and the exon 21 primer was treated with XhoI in exon 16 was treated. Was cloned from the 3'-arm of exon 16-intron 21 using an exon 16 primer and a primer added with an XbaI recognition sequence at intron 21 site (Table 1). PBS-HSVTK-PGKNeo (Professor Koichi Ikuta, Institute for Virus Research, Kyoto University) The FVIII gene targeting vector was prepared by inserting PGK-Neo into exon 16 and placing HSV-TK, which is a suicide gene, upstream of the 5′-arm. For use in electroporation, it was linearized by digestion with NotI (TOYOBO), purified with phenol / chloroform, and diluted with HBS (HEPEs Buffered Saline) to a concentration of 25 nM.
２．制限酵素地図の作成
５’−アームあるいは３’−アームをクローニングしたプラスミドを任意の制限酵素にて３７℃、２時間処理後、アガロース電気泳動とエチジウムブロマイド染色にて消化バンドを確認し、ＤＮＡ塩基配列決定した配列の制限酵素認識部位を照合させ、制限酵素地図を作成した。
2. Preparation of restriction enzyme map After 5'-arm or 3'-arm cloned plasmid was treated with any restriction enzyme at 37 ° C for 2 hours, digestion band was confirmed by agarose electrophoresis and ethidium bromide staining, DNA base A restriction enzyme map was prepared by collating the restriction enzyme recognition sites of the sequence determined.
３．胎児線維芽細胞の採取
ランドレース♀×大ヨークシャー♂で交配後、妊娠６５もしくは７２日目の胎児を採取した。採取した胎児は、ハサミで筋肉組織を採取し、細切した。細切した胎児組織１ｇ当たり１０ｍｌの０．２５％トリプシン−０．０４％ＥＤＴＡ−２Ｎａ液に懸濁し、一晩冷蔵した（４℃）。遠心処理（１５００ｒｐｍ、５ｍｉｎ）により上清を除去した後、３７℃に保温したウォーターバスにて２０−３０分間処理した。保温処理後、胎児組織１ｇ当たり１０ｍｌのＤＭＥＭ＋１０％ＦＣＳを加えてピペッティングし、セルストイレーナーにて濾過した。濾過液を遠心後、上清を除去し、沈殿物に１０ｍｌのＤＭＥＭ＋１０％ＦＣＳを添加して再懸濁した。細胞数を計測し、適当量になるようＤＭＥＭ＋１０％ＦＣＳにて調整した後、７５ｃｍ２細胞培養用フラスコに播いた。その播いた細胞は、３７℃で５％ＣＯ２でコンフルエントになるまで培養し、以下のエレクトロポレーションによる遺伝子導入に用いた。
3. Collection of fetal fibroblasts After mating with Landrace ♀ × Large Yorkshire 胎 児, fetuses at 65 or 72 days of gestation were collected. For the collected fetus, muscle tissue was collected with scissors and minced. The suspension was suspended in 10 ml of 0.25% trypsin-0.04% EDTA-2Na solution per 1 g of minced fetal tissue and refrigerated overnight (4 ° C.). After removing the supernatant by centrifugation (1500 rpm, 5 min), it was treated in a water bath kept at 37 ° C. for 20-30 minutes. After the heat treatment, 10 ml of DMEM + 10% FCS per 1 g of fetal tissue was added and pipetted, followed by filtration with a Celes toilet. After centrifuging the filtrate, the supernatant was removed, and 10 ml of DMEM + 10% FCS was added to the precipitate and resuspended. The number of cells was counted, adjusted to an appropriate amount with DMEM + 10% FCS, and then seeded in a 75 cm 2 cell culture flask. The seeded cells were cultured at 37 ° C. with 5% CO 2 until they became confluent and used for gene transfer by the following electroporation.
４．遺伝子導入とＧ４１８薬剤選択（ＦVIII遺伝子ＫＯ細胞の作出）
分離および増殖した胎児線維芽細胞は、０．２５％トリプシン−０．０４％ＥＤＴＡ−２Ｎａ液で剥離させ、ＤＭＥＭ＋１０％ＦＣＳにて懸濁した。細胞懸濁液を遠心処理（１５００ｒｐｍ、５ｍｉｎ）した後、上清を除去し、冷ＨＢＳに再懸濁した。再懸濁液は、再度遠心処理を行って上清を除去した。細胞数が２．５×１０７細胞／ｍｌになるようにＨＢＳにて調整した。キュベットに細胞溶液０．４ｍｌ（１．０×１０７細胞／ｍｌ）と１００μｌの調整済みＤＮＡ液（２５ｎＭの直線化したターゲッティングベクターを添加、図１．）を加えた後、良く混和した。遺伝子導入には、電気穿孔法装置を３００Ｖ、９５０μＦ、抵抗∞の条件のパルスにて行った。パルス付加後、１０分間室温にて静置した。静置した細胞は、ＤＭＥＭ＋１０％ＦＣＳに懸濁後、３枚の１０ｃｍシャーレに分けて培養した。パルス後４８時間目に８００μｇ／ｍｌのネオマイシン（Ｇ４１８）および２μＭガンシクロビア（Denosine；Mitsubishi Tanabe Pharma社製）を培養液に添加し、耐性細胞株を７−１２日間選択培養した。薬剤選択後、ＰＢＳで洗浄し、０．２５％トリプシン−ＥＤＴＡ（Invitrogen社製）をしみ込ませたペーパーディスク（ADVANTEC社製）を細胞コロニーに乗せ、３７℃で３分インキュベーションした後、培地を入れておいた２４ウェル-プレート（Corning社製）へ１枚／ウェルとなるように継代した。培養５−６日目にフルコンフルエントになった各コロニーは１／６ずつ２４ウェル−プレートの３ウェルに継代し、１／２の細胞についてゲノムＤＮＡを抽出し、相同組換えにより生じた遺伝子組換え体についてスクリーニングした。同様に継代した３ウェルについてもフルコンフルエントにした後に細胞継代とゲノムＤＮＡを抽出し、確認のＰＣＲスクリーニングをした。ＰＣＲスクリーニングで陽性の結果が出たコロニーは、そのまま培養を継続し、核移植に供した。
4). Gene transfer and G418 drug selection (FVIII gene KO cell production)
The separated and expanded fetal fibroblasts were detached with 0.25% trypsin-0.04% EDTA-2Na solution and suspended in DMEM + 10% FCS. After the cell suspension was centrifuged (1500 rpm, 5 min), the supernatant was removed and resuspended in cold HBS. The resuspension was centrifuged again to remove the supernatant. The cells were adjusted with HBS so that the number of cells became 2.5 × 10 7 cells / ml. The cell solution 0.4 ml (1.0 × 10 7 cells / ml) and 100 μl of the prepared DNA solution (25 nM linearized targeting vector was added, FIG. 1) were added to the cuvette and mixed well. For gene transfer, an electroporation apparatus was used with pulses of 300 V, 950 μF, and resistance ∞. After applying the pulse, it was allowed to stand at room temperature for 10 minutes. The stationary cells were suspended in DMEM + 10% FCS, and then divided into three 10 cm dishes and cultured. At 48 hours after the pulse, 800 μg / ml neomycin (G418) and 2 μM gancyclovir (Denosine; manufactured by Mitsubishi Tanabe Pharma) were added to the culture solution, and the resistant cell line was selectively cultured for 7 to 12 days. After drug selection, wash with PBS, place a paper disk (ADVANTEC) soaked with 0.25% trypsin-EDTA (Invitrogen) on the cell colony, incubate at 37 ° C for 3 minutes, and then add the medium. The cells were subcultured at a rate of 1 / well onto a 24-well plate (Corning). Each colony that became fully confluent on the 5th to 6th day of culture was passaged 1/6 to 24 wells-3 wells of a plate, genomic DNA was extracted from 1/2 cell, and the gene produced by homologous recombination Recombinants were screened. Similarly, 3 wells passaged were also made fully confluent and then cell passage and genomic DNA were extracted and subjected to confirmation PCR screening. The colonies that gave a positive result in the PCR screening were continuously cultured and subjected to nuclear transfer.
５．ＰＣＲ解析によるターゲッティングの検出
ＰＣＲスクリーニングはエキソン１４とエキソン１８に設置したプライマー（表２）を用いたＰＣＲによりＦVIII遺伝子組み換え体コロニーを特定し、Ｎｅｏに設置したプライマー（表２）とのＰＣＲにより確認した。また、Ｎｅｏ遺伝子の挿入を確かめるため、エキソン１４―１８のＰＣＲで得られたバンドをＮｅｏ遺伝子に存在するStuIの消化により確認した。ＦVIII遺伝子組換え体コロニーはＧ０期へ以降するまで増殖させ、核移植に使用した。核移植・胚移植由来の雄ブタ胎仔ゲノムについても同様のＰＣＲにより解析した。細胞は、２００ｇ／ｍｌのプロテナーゼＫが含む５０μｌの超純水に再懸濁し、５５℃で３時間保温した後、９５℃１０分間加熱してプロテナーゼを失活させた。ＰＣＲ分析は、Roche社製Expand Long Template PCR Systemを用いた。２５μｌ量の反応液中に消化した５０μｌのサンプルから鋳型として１μｌ（３０〜５００ｎｇ／μｌ）を用いた。ＰＣＲサイクルのパラメーターは、最初に９４℃を２分間、次に９４℃を１０秒、６８℃を６分の１サイクルを１０サイクル、９４℃を１５秒、６８℃を６分の１サイクルにおいて６８℃については、１サイクル毎に２０秒間増加させ２５サイクル後、６８℃を７分間処理した。プライマーはＦ８エキソン１４Ｆ（５’−ＣＡＴＣＡＧＡＧＧＧＡＣＡＴＡＡＧＣＣＴＴＣＣＴＡＣ−３’）およびＦ８エキソン１８Ｒ（５’−ＧＣＣＡＧＧＧＡＧＴＧＴＡＴＣＣＡＴＣＡＣＡＴＡＧ−３’）を用いた。このプライマーは、８ｋｂの変異アレル特異的な産物を設定した。精製したＤＮＡに対するＰＣＲは、鋳型として１００ｎｇの精製ＤＮＡを使用した点を除いて同様に実施した。
5. Detection of targeting by PCR analysis PCR screening identifies FVIII gene recombinant colonies by PCR using primers (Table 2) installed in exon 14 and exon 18, and confirms by PCR with primers installed in Neo (Table 2) did. In addition, in order to confirm the insertion of the Neo gene, the band obtained by PCR of exon 14-18 was confirmed by digestion of StuI present in the Neo gene. FVIII gene recombinant colonies were grown until the GO phase and used for nuclear transfer. The same bovine fetal genome derived from nuclear transfer / embryo transfer was analyzed by the same PCR. The cells were resuspended in 50 μl of ultrapure water containing 200 g / ml of proteinase K, incubated at 55 ° C. for 3 hours, and then heated at 95 ° C. for 10 minutes to deactivate the proteinase. For PCR analysis, an Expand Long Template PCR System manufactured by Roche was used. 1 μl (30 to 500 ng / μl) was used as a template from a 50 μl sample digested in a 25 μl reaction volume. The PCR cycle parameters were as follows: 94 ° C for 2 minutes, then 94 ° C for 10 seconds, 68 ° C for 1/6 cycle, 94 ° C for 15 seconds, 68 ° C for 1/6 cycle 68 About 25 degreeC, it increased for 20 second for every cycle, and after 25 cycles, 68 degreeC was processed for 7 minutes. As the primers, F8 exon 14F (5′-CATCAGAGGGACATAAGCCTTCCTAC-3 ′) and F8 exon 18R (5′-GCCAGGGAGTGTATTCCATCACATAG-3 ′) were used. This primer set an 8 kb mutant allele specific product. PCR on the purified DNA was performed in the same manner except that 100 ng of purified DNA was used as a template.
６．ホルモン処理による採卵方法
ランドレース（Ｌ）およびランドレース、大ヨークシャー（Ｗ）、デュロック（Ｄ）の三元交雑種（ＬＷＤ）の生後１６０〜１９４日齢の春期発動前の雌ブタを用いた。供試ブタにｅＣＧ（ピーメックス、三共）を１５００ＩＵ投与し、その７２時間後にｈＣＧ（プペローゲン、三共）を５００ＩＵ投与した。ｈＣＧ投与４８時間後に屠殺し、卵巣、卵管、子宮の結合組織を摘出した。また、性成熟に達した雌豚からの採卵は、予め発情時に雄希釈精液を用いて人工授精を行い、妊娠２１−５０日後に流産処置し行った。核移植予定５日前にＰＧＦ２α類縁体クロプロステロール約０．２ｍｇ（プラネート：２ｍｌ量、武田製薬社製）を臀部筋肉内に投与した。その２４時間後に、同様にＰＧＦ２α類縁体クロプロステロールを注射し、同時にｅＣＧ（ピーメックス、三共製薬社製）を臀部筋肉内に１５００ＩＵ投与した。ＰＧＦ２α＋ｅＣＧ投与７２時間後にｈＣＧ（プペローゲン、三共製薬社製）を５００ＩＵ投与した。ｈＣＧ投与４６時間後に屠殺し、卵巣、卵管、子宮の結合組織を摘出した。
6). Egg-harvesting method by hormonal treatment The sows before the start of spring 160-194 days of age of Landrace (L), Landrace, Large Yorkshire (W), Duroc (D) ternary hybrid (LWD) were used. The test pigs were administered 1500 IU of eCG (Pimex, Sankyo) and 72 hours later, 500 IU of hCG (Puperogen, Sankyo). At 48 hours after hCG administration, the mice were sacrificed, and connective tissues of the ovaries, fallopian tubes, and uterus were removed. In addition, eggs collected from sows that reached sexual maturity were subjected to artificial insemination using male-diluted semen at the time of estrus, and miscarriage treatment was performed 21-50 days after pregnancy. Five days before the planned nuclear transfer, about 0.2 mg of PGF2α analog cloprosterol (planate: 2 ml, Takeda Pharmaceutical Co., Ltd.) was administered intramuscularly. Twenty-four hours later, PGF2α analog cloprosterol was similarly injected, and at the same time, eCG (Pimex, Sankyo Pharmaceutical Co., Ltd.) was administered into the buttocks muscle at 1500 IU. 72 hours after administration of PGF2α + eCG, 500 IU of hCG (Puperogen, Sankyo Pharmaceutical Co., Ltd.) was administered. The mice were sacrificed 46 hours after the administration of hCG, and connective tissues of the ovaries, fallopian tubes and uterus were removed.
７．体内成熟卵子の準備
摘出した卵巣、卵管、子宮の結合組織を直ちに実験室に持ち帰り、灌流液（ＰＢＳ：Ｃａ２＋、Ｍｇ２＋不含、（TAKARA社製）＋０．１％ウシ血清アルブミン：ＢＳＡ（Sigma社製、Ｃａｔ．Ｎｏ．Ａ−４５０３）＋１００ｍｌあたり１ｍｌ添加抗生物質：Antibiotic,Antimycotic（Sigma社製、Ａ−７２９２）で卵管を灌流し、卵子を採取した。得られた卵子は０．１％ヒアルロニダーゼを含む、灌流液を用いて卵丘細胞を裸化し、卵細胞質の均一な卵子を選抜した後、ヒアルロニダーゼを含まない灌流液中で洗浄した。その後、ＰＺＭ−３液（Biol Reprod. 66:112-119. 2002）を用いてインキュベーター（３８．５℃、５％ＣＯ２、５％Ｏ２、９０％Ｎ２）内で供試するまで体外培養を行った。
7). Preparation of mature oocytes in the body The removed connective tissue of the ovary, fallopian tube, and uterus was immediately brought back to the laboratory, and the perfusate (PBS: Ca 2+ , Mg 2+ free (TAKARA) + 0.1% bovine serum albumin: BSA (Sigma, Cat. No. A-4503) + 1 ml added per 100 ml Antibiotic: Antibiotic, Antithycotic (Sigma, A-7292) perfused the oviduct and collected the ovum. The cumulus cells were naked using a perfusate containing 1% hyaluronidase, and eggs with uniform egg cytoplasm were selected and then washed in a perfusate containing no hyaluronidase, followed by PZM-3 solution (Biol Reprod). 66: 112-119. 2002), in vitro culture was performed until it was tested in an incubator (38.5 ° C., 5% CO 2 , 5% O 2 , 90% N 2 ).
８．屠場卵巣由来卵胞卵子の採卵方法
屠場にて食肉用として屠殺された春機発動前の未経産雌ブタより卵巣を採取し、ＰＢＳ（−）液にて３５℃保温下で研究室に持ち帰った。持ち帰った卵巣は直ちに、３７℃に保温したＰＢＳ（−）にて洗浄し、卵巣結合組織を除去した後、採卵作業を行った。直径３−６ｍｍの卵胞を選び、１６Ｇ注射針付き１０ｍｌシリンジで吸引した。吸引した卵胞液は、５０ｍｌディスポチューブに回収し、５分以上室温放置後、上清を除去した。卵子が含まれる沈殿物に対し、ＴＡＬＰ−Ｈｅｐｅｓ液を加えて懸濁後、沈殿物がチューブの底に溜まったのを確認し、沈殿物を吸引しないよう、パスツールピペットにて上清を除去した。この洗浄操作を２−３回繰り返した。卵丘細胞が３層以上密に付着した卵丘細胞卵子複合体（ＣＯＣ）とＣＯＣに壁側顆粒層細胞が付着した壁側顆粒層細胞−卵丘細胞卵子複合体（ＧＣＯＣ）を選別・採卵をした。
8). Ovulation method of ovarian follicular ovum derived from slaughterhouse ovaries were collected from heifers before spring trigger that were slaughtered for meat at the slaughterhouse, and brought back to the laboratory with PBS (−) solution under a temperature of 35 ° C. . The ovaries brought home were immediately washed with PBS (−) kept at 37 ° C., and ovarian connective tissue was removed, and then egg collection was performed. Follicles with a diameter of 3-6 mm were selected and aspirated with a 10 ml syringe with a 16G needle. The aspirated follicular fluid was collected in a 50 ml disposable tube, allowed to stand at room temperature for 5 minutes or more, and then the supernatant was removed. Add suspension of TALP-Hepes to the precipitate containing the egg, confirm that the precipitate has accumulated at the bottom of the tube, and remove the supernatant with a Pasteur pipette so as not to aspirate the precipitate. did. This washing operation was repeated 2-3 times. Selection and egg collection of cumulus cell ovum complex (COC) in which cumulus cells are densely attached in three or more layers and wall side granule layer cell-cumulus cell ovum complex (GCOC) in which wall side granule layer cells are attached to COC Did.
９．体外成熟培養
採取したＣＯＣおよびＧＣＯＣは、約１５〜２０個／穴で改変ＮＣＳＵ３７を１００μｌ分注し、１００μｌ流動パラフィンオイルカバーした６穴ディッシュを用いて成熟培養した。改変ＮＣＳＵ−３７は、１０％ブタ卵胞液、０．６ｍＭシステイン、５０μＭ βメルカプトエタノール、１ｍＭジブチリルｃＡＭＰ、１０ＩＵ／ｍｌ ＰＭＳＧ（ピーメックス；１０００ＩＵ三共製薬社製）、１０ＩＵ／ｍｌ ｈＣＧ（プペローゲン；１５００ＩＵ、三共製薬社製）を添加して作製した。２０〜２２時間まで培養後、ホルモン及びジブチリルｃＡＭＰ無添加改変ＮＣＳＵ３７に移して供試時まで培養した。成熟培養は３９℃、気相条件は、ＣＯ２５％、Ｏ２５％、Ｎ２９０％の条件で実施した。成熟培養時間は、４２−４４時間で行い、成熟卵子（第一極体放出卵子）のみを試験に使用した。
9. In vitro maturation culture The collected COC and GCOC were dispensed 100 μl of modified NCSU37 at about 15 to 20 per well, and matured using a 6-well dish covered with 100 μl of liquid paraffin oil. Modified NCSU-37 is 10% porcine follicular fluid, 0.6 mM cysteine, 50 μM β-mercaptoethanol, 1 mM dibutyryl cAMP, 10 IU / ml PMSG (Pimex; manufactured by 1000 IU Sankyo Pharmaceutical Co., Ltd.), 10 IU / ml hCG (Properogen; 1500 IU, (Manufactured by Sankyo Pharmaceutical Co., Ltd.). After culturing for 20 to 22 hours, it was transferred to a modified NCSU 37 without hormones and dibutyryl cAMP and cultured until the test. The mature culture was performed at 39 ° C., and the gas phase conditions were CO 2 5%, O 2 5%, and N 2 90%. The mature culture time was 42-44 hours, and only mature eggs (first polar body released eggs) were used for the test.
１０．卵子核の除去操作（除核操作）
除核操作前に、屠殺採卵した未受精卵子を、サイトカラシンＢ（Sigma社製：Ｃａｔ．Ｎｏ．Ｃ−６７６２）が５μｇ／ｍｌ濃度含むＰＺＭ３液に移し、１５分間以上処理した。除核操作も同濃度のサイトカラシンＢが含まれるＰＺＭ３液で作製したドロップ内（１０ｃｍプラスチックシャーレの中心付近に５０μｌでドロップを作製し、ミネラルオイル２０ｍｌで被った）で行った。卵子は、第一極体が、１２時から３時方向の位置になるようにホールディングピペットで保定した。ピエゾマイクロマニピュレーター（プライムテック株式会社製）に取り付けた除核用ピペット（外径２５〜３０μｍ、先端を３０〜４５度の角度で研磨）により、第一極体ごと細胞質を１／４〜１／３量程吸引した。除核用ピペットの操作性が悪くなった場合は、２０％ＰＶＰ液（Sigma社製：Ｃａｔ．Ｎｏ．ＰＶＰ−３６０）ドロップにて、吸引・排出を繰り返し、洗浄を行った。除核操作終了後、直ちにサイトカラシンＢが含まれないＰＺＭ３液に移し、洗浄を行う。洗浄には、ＰＺＭ３液が３ｍｌ入った３５ｍｍディッシュ（Falcon社製、１００８）で２回以上、丁寧に洗浄し、サイトカラシンＢを除去した。その後、注入操作まで、ＰＺＭ３液ドロップ（３５ｍｍディッシュ：Falcon社製、３００１に１００μｌのドロップを作製し、４ｍｌミネラルオイルで被った）に移し、インキュベーター内で培養した。
10. Oocyte nucleus removal operation (enucleation operation)
Prior to the enucleation operation, the slaughtered unfertilized ovum was transferred to a PZM3 solution containing 5 μg / ml of cytochalasin B (Sigma: Cat. No. C-6762) and treated for 15 minutes or more. The enucleation operation was also carried out in a drop made with PZM3 solution containing cytochalasin B at the same concentration (prepared with 50 μl of a drop near the center of a 10 cm plastic petri dish and covered with 20 ml of mineral oil). The egg was held with a holding pipette so that the first polar body was positioned from 12:00 to 3 o'clock. The cytoplasm of the first polar body is reduced to 1/4 to 1/1 / by a pipette for enucleation (outer diameter 25 to 30 μm, the tip polished at an angle of 30 to 45 degrees) attached to a piezo micromanipulator (manufactured by Primetec Corporation). Aspirate about 3 volumes. When the operability of the enucleating pipette deteriorated, washing was performed by repeatedly sucking and discharging with a 20% PVP solution (Sigma: Cat. No. PVP-360) drop. Immediately after the enucleation operation is completed, transfer to a PZM3 solution containing no cytochalasin B and perform washing. For washing, cytochalasin B was removed by washing carefully with a 35 mm dish (manufactured by Falcon, 1008) containing 3 ml of PZM3 solution twice or more. Thereafter, until injection operation, the solution was transferred to a PZM 3 liquid drop (35 mm dish: manufactured by Falcon, 1001 drop on 3001, covered with 4 ml mineral oil) and cultured in an incubator.
１１．ドナー組換え体細胞の準備
ＰＣＲ解析によりＦVIII遺伝子がノックアウトされていると判定した雄胎児の線維芽細胞を核移植ドナー細胞として用いた。培養液にはＤＭＥＭ＋１０％ ＦＣＳを用い、植え継ぎ回数は０〜５回の細胞を用いた。核移植予定日に合わせて、植え継ぎを行った後、コンフルエント状態から培養液の交換を行わず、３〜１４日間放置し、細胞周期をＧ１／Ｇ０期に同調した。核移植に用いる約１時間前にドナー体細胞の準備を行った。細胞処理は、培養容器の培養液を除去した後に、ＰＢＳ（−）にて３回以上洗浄を行い、０．２５％トリプシン＋０．０４％ＥＤＴＡ-２Ｎａ液にて細胞剥離処理を行った。剥離した細胞に１０％ＦＣＳ添加ＤＭＥＭを加えて懸濁した後、遠心処理（１，５００ｒｐｍ、５ｍｉｎ、室温）を行った。遠心処理後、上清を除去し、ＰＺＭ３液を適量加えて懸濁し、核移植に用いるまで室温で放置した。体細胞核注入操作時に、適当な細胞数を注入操作用チャンバーのドロップに添加して使用した。
11. Preparation of Donor Recombinant Cells Male fetal fibroblasts determined to have been knocked out by FVIII gene by PCR analysis were used as nuclear transfer donor cells. As the culture solution, DMEM + 10% FCS was used, and cells were transplanted 0 to 5 times. In accordance with the scheduled day of nuclear transfer, after transplanting, the culture medium was not changed from the confluent state and left for 3 to 14 days to synchronize the cell cycle with the G1 / G0 phase. Donor somatic cells were prepared approximately 1 hour before being used for nuclear transfer. In the cell treatment, after removing the culture solution from the culture vessel, washing was performed 3 times or more with PBS (−), and cell detachment treatment was performed with 0.25% trypsin + 0.04% EDTA-2Na solution. The detached cells were suspended by adding DMEM supplemented with 10% FCS, followed by centrifugation (1,500 rpm, 5 min, room temperature). After centrifugation, the supernatant was removed, an appropriate amount of PZM3 solution was added, suspended, and left at room temperature until used for nuclear transfer. At the time of somatic cell nucleus injection operation, an appropriate number of cells was added to the drop of the injection operation chamber for use.
１２．体細胞核の注入操作
注入用チャンバー（１０ｃｍシャーレにＰＺＭ３液５０μｌでドロップを作製し、ミネラルオイル２０ｍｌで被った）のドロップに適量のドナー細胞を添加し、除核済み卵子５０〜１００個を入れて操作した。注入操作には、ピエゾマイクロマニュピレーターに体細胞核注入用ピペット（外径７−１０μｍ、鈍端）を用いた。注入用ピペットをピエゾマイクロマニュピレーターに取り付ける前に、ピペット後端より５ｍｍの位置にフロリナートを３−５ｍｍ幅になるように、充填して使用した。そのピペットをピエゾに取り付けて使用する時は、先端より培養液、フロリナート、空気、ミネラルオイルの順で満たされている状態にして操作した。注入操作を行う前に、必ず２０％ＰＶＰ液ドロップ内で注入用ピペットを洗浄（ピペッティングやＰＶＰドロップへのピペット先端の出し入れ）した。浮遊している体細胞を吸引し、数回ピペッティングを行い、細胞膜を壊し、ホールディングピペットにて保定した除核済み卵子細胞質内へ注入した。体細胞核を注入した卵子は、活性化処理時間までインキュベーターで培養を行った（１〜３時間）。
12 Somatic cell nucleus injection operation Add an appropriate amount of donor cells to a drop of an injection chamber (prepared with 50 μl of PZM3 solution in a 10 cm petri dish and covered with 20 ml of mineral oil), and put 50-100 enucleated eggs. Operated. For the injection operation, a pipette for somatic cell nucleus injection (outer diameter 7-10 μm, blunt end) was used in a piezo micromanipulator. Before the injection pipette was attached to the piezo micromanipulator, it was used by filling Fluorinert with a width of 3-5 mm at a position 5 mm from the rear end of the pipette. When using the pipette attached to the piezo, the pipette was filled with culture solution, florinate, air, and mineral oil in that order. Before performing the injection operation, the injection pipette was always washed in the 20% PVP liquid drop (pipetting and the pipette tip into and out of the PVP drop). The floating somatic cells were aspirated and pipetted several times to break the cell membrane and injected into the enucleated oocyte cytoplasm held with a holding pipette. Oocytes injected with somatic cell nuclei were cultured in an incubator until the activation treatment time (1 to 3 hours).
１３．卵子の活性化処理
体細胞核を注入した卵子の活性化処理は直流パルス刺激（ＳＳＨ−２、島津製作所製）を、１．５ｋＶ／ｃｍ １００μｓｅｃ×１回の条件にて行った。チャンバーには２ｍｍ幅のステンレスワイヤー電極を用い、電気刺激用の電解質溶液には０．０５ｍＭ ＣａＣｌ２、０．１ｍＭ ＭｇＳＯ４ならびに０．０２％ＢＳＡを含む０．２８Ｍマンニトール溶液を用いた。
13. Ovum Activation Treatment The activation treatment of eggs injected with somatic cell nuclei was performed by direct current pulse stimulation (SSH-2, manufactured by Shimadzu Corporation) under the conditions of 1.5 kV / cm 100 μsec × 1 time. A stainless steel wire electrode having a width of 2 mm was used for the chamber, and a 0.28 M mannitol solution containing 0.05 mM CaCl 2 , 0.1 mM MgSO 4 and 0.02% BSA was used for the electrolyte solution for electrical stimulation.
１４．活性化卵子の第２極体放出抑制処理
活性化処理卵子は５μｇ／ｍｌサイトカラシンＢ（Sigma社製：Ｃａｔ．Ｎｏ．Ｃ−６７６２）を含むＰＺＭ３液で２時間培養し、第２極体の放出を抑制させる処理を行った。
14 Second Polar Body Release Suppression Treatment of Activated Egg The activated egg was cultured in PZM3 solution containing 5 μg / ml cytochalasin B (Sigma: Cat. No. C-6762) for 2 hours. A treatment to suppress the release was performed.
１５．活性化卵子の体外培養
活性化後２倍体処理をした卵子は、ＰＺＭ３液にて体外培養を行い、活性化処理後２日目に分割胚のみ胚移植に用いた。
15. In Vitro Culture of Activated Ovum Eggs that had been diploid treated after activation were subjected to in vitro culture in PZM3 solution, and only split embryos were used for embryo transfer on the second day after the activation treatment.
１６．ホルモン処理による仮腹の同期化と胚移植方法
仮腹の同期化は妊娠豚を人工流産処置することで行った。また、仮腹の発情は、採卵豚のものより、１日遅れるように発情同期化処置を行った。供試予定の雌ブタは、発情時に、雄豚希釈精液を用いて人工授精を行い妊娠させた。妊娠後２１−５０日目の妊娠豚を用いた。核移植予定６日前にＰＧＦ２α類縁体クロプロステロール約０．２ｍｇ（プラネート、武田製薬社製：２ｍｌ量）を臀部に注射した。その２４時間後に、同様にＰＧＦ２α類縁体クロプロステロールを注射し、同時にｅＣＧ（ピーメックス、三共製薬社製）を１０００ＩＵ投与した。ＰＧＦ２α＋ｅＣＧ投与７２時間後にｈＣＧ（プペローゲン、三共製薬社製）を５００ＩＵ投与した。ｈＣＧ投与、約６８時間目に仮腹へケタラール投与後、ハロセン麻酔下で外科的に胚移植手術を行った。胚移植はＰＰカテーテル（フジヒラ社製）を卵管へ挿入して行った。
16. Synchronization of temporary stomach by hormone treatment and embryo transfer method Synchronization of temporary stomach was performed by artificial abortion treatment of pregnant pigs. Further, the estrus synchronization treatment was performed so that the estrus of the provisional stomach was delayed one day from that of the egg-collecting pig. The sows to be tested were made pregnant by artificial insemination using boar diluted semen at the time of estrus. Pregnant pigs 21-50 days after pregnancy were used. Six days before the planned nuclear transfer, about 0.2 mg of PGF2α analog cloprosterol (Planate, Takeda Pharmaceutical Co., Ltd .: 2 ml amount) was injected into the buttocks. After 24 hours, PGF2α analog cloprosterol was similarly injected, and at the same time, eCG (Pimex, Sankyo Pharmaceutical Co., Ltd.) was administered at 1000 IU. 72 hours after administration of PGF2α + eCG, 500 IU of hCG (Puperogen, Sankyo Pharmaceutical Co., Ltd.) was administered. At about 68 hours after hCG administration, ketalal was administered to the abdomen, and then embryo transfer was performed surgically under halothane anesthesia. Embryo transfer was performed by inserting a PP catheter (Fujihira) into the fallopian tube.
１７．組み換えクローン胎児の採取および細胞分離作業
同定したＰＣＲ陽性コロニーは、非相同組換え細胞が、様々な割合で含まれている可能性があり、体細胞核移植技術を用いた効率的な相同組換え動物の作出の妨げとなりうることが予想された。そこで、単一な相同組換え細胞のみの集団を得るため、妊娠した雌ブタの３２日目もしくは３９日目に胎児を採取した。採取した胎児は、３．と同様に細胞を分離すると同時にゲノムＤＮＡを採取し、相同組み換え個体であるか５．と同様の条件でＰＣＲ解析した。ＰＣＲ解析にて陽性であった胎児細胞を用いて再核移植を実施し、胚移植によりＦVIIIノックアウトクローン個体の作出に供した。
17. Recombinant cloned fetus collection and cell separation work The identified PCR-positive colonies may contain non-homologous recombination cells in various proportions. Efficient homologous recombination animals using somatic cell nuclear transfer technology It was expected that it could hinder the creation of Therefore, in order to obtain a population of only single homologous recombinant cells, fetuses were collected on the 32nd or 39th day of pregnant sows. The collected fetus is 3. 4. Isolate cells in the same manner as above, collect genomic DNA at the same time, and are homologous recombination individuals? PCR analysis was performed under the same conditions. Renuclear transfer was performed using fetal cells that were positive in PCR analysis, and FVIII knockout clones were produced by embryo transfer.
１８．サザンブロット解析
核移植・胚移植により発生した雄ブタ胎仔線維芽細胞から抽出したゲノムＤＮＡを用いてＦVIII遺伝子の組換えをサザンブロットにて確認した。５ｍｇのゲノムＤＮＡを制限酵素SacI、StuI、EcoRI、SphI、あるいはXbaIで切断し、アガロース電気泳動後、アルカリトランスファー条件によるサザンブロッティングを実施した。バンドの検出はDIG PCR Labeling Kit（Roche社製）にてエキソン１４またはエキソン２２のＤＩＧラベルプローブを、エキソンプライマー（表３）を用いたＰＣＲにより作製し、DIG Easy Hyb（Roche社製）にてハイブリダイゼーション、DIG Detection Kit（Roche社製）により検出した。
18. Southern blot analysis FVIII gene recombination was confirmed by Southern blotting using genomic DNA extracted from bovine fetal fibroblasts generated by nuclear transfer / embryo transfer. 5 mg of genomic DNA was cleaved with restriction enzymes SacI, StuI, EcoRI, SphI or XbaI, subjected to Southern blotting under alkaline transfer conditions after agarose electrophoresis. For detection of the band, a DIG label probe of exon 14 or exon 22 was prepared by PCR using an exon primer (Table 3) with DIG PCR Labeling Kit (Roche), and DIG Easy Hyb (Roche) was used. Hybridization was detected by DIG Detection Kit (Roche).
１９．ブタ血漿の解析
血液と３．８％クエン酸ナトリウム溶液（Harasawa Pharmaceutical社製）を１／１０となるようにブタから採血し、３０００ｒｐｍ、１０分の遠心分離により血漿を分離した。活性化部分トロンボプラスチン時間（ＡＰＴＴ）をCA500（Sysmex社製） にて測定し、ヒトＦVIIIに対する抗体Sheep anti-human FVIII Antibody（Affinity Biologicals Inc.社製）とRabbit Anti-human FVIII（研究室で作製したポリクローナル抗体）、Goat anti-Rabbit IgG-HRP（BioSourse社製）を用いたＥＬＩＳＡにより、ＦVIIIの検出を検討した。ヒト正常血漿（健常者ボランティアによる）においてＡＰＴＴのスタンダードを作製し、ヒト血漿ＦVIII活性を１００％としてブタ血漿ＦVIII活性を算出した。ＥＬＩＳＡでは、ブタ血漿とヒト血漿、それらの混和（ブタ／ヒト：１／１〜１／５０）をサンプルとし、Benchmark plus（Bio-Rad Laboratories社製）にて吸光度４１５ｎｍを測定し、ＡＢＴＳ（Kirkegaard＆Perry Laboratories社製）の発色によりＦVIIIを検出した。
19. Analysis of Porcine Plasma Blood and 3.8% sodium citrate solution (Harasawa Pharmaceutical Co., Ltd.) were collected from the pig so as to be 1/10, and plasma was separated by centrifugation at 3000 rpm for 10 minutes. Activated partial thromboplastin time (APTT) was measured with CA500 (manufactured by Sysmex) and antibodies against human FVIII, Sheep anti-human FVIII antibody (manufactured by Affinity Biologicals Inc.) and Rabbit Anti-human FVIII (manufactured in the laboratory) The detection of FVIII was examined by ELISA using a polyclonal antibody) and Goat anti-Rabbit IgG-HRP (BioSourse). A standard for APTT was prepared in human normal plasma (from healthy volunteers) and porcine plasma FVIII activity was calculated with human plasma FVIII activity as 100%. In ELISA, porcine plasma and human plasma, and their mixture (pig / human: 1/1 to 1/50) are used as samples, the absorbance at 415 nm is measured with Benchmark plus (manufactured by Bio-Rad Laboratories), and ABTS (Kirkegaard & Perry) is measured. FVIII was detected by color development of (Laboratories).
２０．誕生したクローンブタのＰＣＲ判定
誕生したクローンブタから採取可能な組織（耳、臍帯組織）や血液等からＤＮＡを抽出し、前記５．「ＰＣＲ解析によるターゲッティングの検出」に準じてＰＣＲ解析を行った。
20. 4. PCR determination of the born cloned pig DNA is extracted from tissue (ear, umbilical cord tissue) or blood that can be collected from the born cloned pig. PCR analysis was performed according to “Detection of targeting by PCR analysis”.
（ＦVIII遺伝子ターゲッティングベクター）
ブタの全ゲノムＤＮＡ配列が解読・公開されていないため、現時点で公開されているブタ肝臓からクローニングされたＦVIII ｃＤＮＡ配列をヒトＦVIII ｃＤＮＡ配列との相同性によりエキソン部位を予測、ＰＣＲにより増幅・クローニングすることで遺伝子発現を欠損させるためのターゲッティングベクターを構築し、配列を解読した。ｃＤＮＡ配列とクローニングしたゲノムＤＮＡ配列の照合によってエキソン部位が特定された。
また、種々の制限酵素処理によってクローニングしたゲノムＤＮＡ領域の制限酵素地図を作製した（図１参照）。
(FVIII gene targeting vector)
Since the whole genome DNA sequence of pig has not been decoded and published, the exon site is predicted by homology with the human FVIII cDNA sequence and the FVIII cDNA sequence cloned from the currently published pig liver is amplified and cloned by PCR Thus, a targeting vector for deleting gene expression was constructed, and the sequence was decoded. Exon sites were identified by matching the cDNA sequence with the cloned genomic DNA sequence.
Moreover, restriction enzyme maps of genomic DNA regions cloned by various restriction enzyme treatments were prepared (see FIG. 1).
（ＦVIII遺伝子組換えブタ胎仔線維芽細胞の作出）
ＦVIII遺伝子ターゲティングベクターの導入と薬剤選択によって得られた９３６コロニーのうち増殖可能であった２００コロニーをＰＣＲスクリーニングした。遺伝子導入の回数と薬剤耐性コロニー数、増殖コロニー数、ＦVIII遺伝子組換え型コロニー数を表４に示す。エキソン１４−１８において、遺伝子組換え型ではエキソン１６へのＮｅｏの挿入により遺伝子増幅部位が長くなることを検証したところ、野生型と遺伝子組換え型の両方にバンドが出るコロニー＃１３４が１つ得られた（図２）。コロニー＃１３４におけるＮｅｏの挿入はＰＣＲ（エキソン１４−Ｎｅｏ、Ｎｅｏ-エキソン２２）とエキソン１４-１８のＰＣＲ後のＳｔｕＩの切断により陽性と確認された（図２）。さらに継代した３ウェルについても同様の結果が得られた（図２）。よって、得られたコロニー＃１３４は野生型細胞とエキソン１６にＮｅｏが挿入されたＦVIII遺伝子組換え細胞とを含む細胞集団であると考えられた。このコロニー＃１３４を核移植に用いた。
(Production of FVIII transgenic pig fetal fibroblasts)
Of the 936 colonies obtained by introduction of the FVIII gene targeting vector and drug selection, 200 colonies that could grow were subjected to PCR screening. Table 4 shows the number of gene introductions, the number of drug-resistant colonies, the number of proliferating colonies, and the number of FVIII gene recombinant colonies. In exon 14-18, when it was verified that the gene amplification site becomes longer due to the insertion of Neo into exon 16 in the gene recombinant type, one colony # 134 in which a band appears in both the wild type and the gene recombinant type Obtained (FIG. 2). Neo insertion in colony # 134 was confirmed to be positive by PCR (exon 14-Neo, Neo-exon 22) and StuI digestion after PCR of exon 14-18 (FIG. 2). Further, similar results were obtained for the subcultured 3 wells (FIG. 2). Therefore, the obtained colony # 134 was considered to be a cell population containing wild type cells and FVIII gene recombinant cells in which Neo was inserted into exon 16. This colony # 134 was used for nuclear transfer.
（ＦVIII遺伝子組換え細胞をもちいた核移植由来胎仔の解析）
ＦVIII遺伝子組換え細胞コロニー＃１３４を用いた計５回の核移植・胚移植の結果、１匹のブタ胎仔が得られ、その線維芽細胞を分離・増殖・保存し、そのゲノムＤＮＡの解析を検討した。ＰＣＲによる解析はエキソン１４−１８において遺伝子組換え型のみを示す結果が得られ、エキソン１６へのＮｅｏの挿入も陽性であった（図３Ａ）。
また、サザンブロッティングによりＦVIII遺伝子の組換えの確認したところ、エキソン１４プローブまたはエキソン２２プローブを用いた場合において、任意のバンドシフトが認められ（図３Ｂ、３Ｃ）、目的であるＦVIII遺伝子のエキソン１６にＮｅｏが挿入されているという結果が得られた。よって、核移植に用いた細胞コロニーはＦVIII遺伝子組換え細胞と野生型細胞が混在する細胞集団であったが、核移植後に発生したクローンブタ胎仔はＦVIII遺伝子組換えブタ（ＦVIII−ＫＯブタ）であることが示された。
(Analysis of embryos derived from nuclear transfer using FVIII transgenic cells)
As a result of 5 times of nuclear transfer / embryo transfer using FVIII gene recombinant cell colony # 134, one pig fetus was obtained, the fibroblasts were isolated, proliferated and stored, and the genomic DNA was analyzed. investigated. As a result of PCR analysis, it was found that exon 14-18 showed only the recombinant type, and insertion of Neo into exon 16 was also positive (FIG. 3A).
Further, when the recombination of the FVIII gene was confirmed by Southern blotting, an arbitrary band shift was observed when the exon 14 probe or the exon 22 probe was used (FIGS. 3B and 3C), and exon 16 of the target FVIII gene was observed. The result that Neo was inserted in was obtained. Therefore, the cell colony used for nuclear transfer was a cell population in which FVIII recombinant cells and wild-type cells were mixed, but the cloned pig fetus generated after nuclear transfer was FVIII recombinant pig (FVIII-KO pig). It was shown that there is.
（ＦVIII遺伝子組換えブタ胎仔細胞の再核移植・胚移植）
作出した細胞由来のクローンブタ胎仔がＦVIII遺伝子組換え（ＦVIII−ＫＯ）ブタであることがゲノムＤＮＡの解析により確かめられたため、そのブタ胎仔線維芽細胞をもちいて計７回の核移植・胚移植を実施した。その結果、３頭の仮親が、妊娠し、分娩まで至った。４頭の生存クローンブタが誕生した。胚移植の実施日程と妊娠の有無、出産予定日、分娩頭数（死産）の結果を表５に示す。
(Renuclear transfer / embryo transfer of FVIII transgenic pig fetal cells)
Since it was confirmed by genomic DNA analysis that the cloned pig fetus derived from the generated cells was FVIII transgenic (FVIII-KO) pig, a total of 7 nuclear transfer / embryo transfer using the porcine fetal fibroblasts Carried out. As a result, three temporary parents became pregnant and delivered. Four surviving clone pigs were born. Table 5 shows the results of the embryo transfer schedule, the presence of pregnancy, the expected date of delivery, and the number of calvings (dead birth).
（ＦVIII−ＫＯブタ新生仔の観察）
ＦVIII−ＫＯ胎仔線維芽細胞の核移植・胚移植により、４頭のＦVIII−ＫＯクローンブタが誕生した。しかし、出産直後あるいは２日目までに３頭が死亡した。
死亡したＦVIII−ＫＯブタ新生仔の剖検から、体の複数箇所に内出血が認められた（図４Ａ）。母ブタとの接触による内出血と推察された。また、内出血部位を切開したところ、血腫が認められ（図４Ｃ）、ＦVIII−ＫＯブタ自身には死亡前に出血傾向があるものと考えられた。
一方、頭蓋内には出血は認められず、臓器所見も異常がなかった（図４Ｂ、４Ｄ）。ＦVIII−ＫＯブタ新生仔のＤＮＡをエキソン１４−１８ ＰＣＲにより解析したところ、２頭ともＦVIII−ＫＯのクローンであることが確認された。
(FVIII-KO pig newborn observation)
Four FVIII-KO cloned pigs were born by nuclear transfer and embryo transfer of FVIII-KO fetal fibroblasts. However, three died immediately after giving birth or by the second day.
From autopsy of the dead FVIII-KO pig newborn, internal hemorrhage was observed in several parts of the body (FIG. 4A). This was presumed to be internal bleeding due to contact with the mother pig. Further, when the internal bleeding site was incised, a hematoma was observed (FIG. 4C), and it was considered that the FVIII-KO pig itself had a bleeding tendency before death.
On the other hand, no bleeding was observed in the cranium, and there were no abnormal organ findings (FIGS. 4B and 4D). Analysis of FVIII-KO newborn pig DNA by exon 14-18 PCR confirmed that both were FVIII-KO clones.
（ブタ血漿の解析）
野生型ブタの血漿をヒトＦVIIIを検出するＥＬＩＳＡによって検出したところ、ブタＦVIIIは検出されず、ヒト血漿との混和によるＥＬＩＳＡにおいてもヒトＦVIIIの検出を阻害するような結果は得られなかった（図５Ａ）。また、野生型ブタ血漿のFVIII活性を測定したところ、ブタ血漿はヒト血漿の約３〜３．５倍のＦVIII活性を有することが示された（図５Ｂ）。
生後２日に出血死したFVIII-ＫＯクローンブタの血液の凝固因子活性（ヒト凝固因子活性換算）を測定したところ、第VIII因子活性は感度以下であり、正常対照となる野生型新生仔ブタの（生後1日）の凝固第VIII因子活性は２００％以上であった。FVIIIＫＯクローンブタの血漿中で低下がみられた第VII因子活性と第XI因子活性は野生型新生仔ブタでも低値であった。FVIII-ＫＯクローンブタでは第VIII因子活性のみが特異的に低下していたことは、得られたクローンブタがFVIII-ＫＯクローンブタであることを確実に示し、出血症状は第VIII因子活性の低下に基づくものであるといえる。
(Analysis of porcine plasma)
When wild-type porcine plasma was detected by ELISA for detecting human FVIII, porcine FVIII was not detected, and even in ELISA by mixing with human plasma, no results were obtained that inhibited the detection of human FVIII (FIG. 5A). Moreover, when FVIII activity of wild-type pig plasma was measured, it was shown that pig plasma has about 3 to 3.5 times as much FVIII activity as human plasma (FIG. 5B).
The blood clotting factor activity (in terms of human clotting factor activity) of the FVIII-KO cloned pig that died of bleeding on the second day after birth was measured. The factor VIII activity was below the sensitivity. The coagulation factor VIII activity (1 day after birth) was 200% or more. Factor VII activity and factor XI activity, which were decreased in plasma of FVIIIKO cloned pigs, were also low in wild-type newborn piglets. The fact that only the factor VIII activity was specifically decreased in the FVIII-KO cloned pigs clearly indicated that the obtained cloned pig was an FVIII-KO cloned pig, and the bleeding symptom was a decrease in the factor VIII activity It can be said that it is based on.
Claims (5)
（ａ）第VIII因子遺伝子のエキソン１６を標的としたターゲティングベクターを作製する工程；
（ｂ）胎児線維芽細胞に、工程（ａ）で作製したターゲティングベクターを導入し、相同組換えにより生じた遺伝子組換え細胞を選抜する工程；
（ｃ）採取した豚の体内成熟卵子から除核する工程；
（ｄ）工程（ｃ）で除核された卵子に、工程（ｃ）で選抜された遺伝子組換え細胞の細胞核を注入する工程；
（ｅ）工程（ｄ）で細胞核が注入された卵子に活性化処理を施し、活性化処理後の核移植胚を雌豚の卵管又は子宮に移植する工程； A method for producing a hemophilia A model pig exhibiting spontaneous bleeding symptoms, comprising the following steps.
(A) producing a targeting vector targeting exon 16 of the factor VIII gene;
(B) introducing the targeting vector prepared in step (a) into fetal fibroblasts and selecting genetically modified cells produced by homologous recombination;
(C) a step of enucleating from the collected porcine mature eggs;
(D) A step of injecting the nuclei of the genetically modified cells selected in the step (c) into the egg enucleated in the step (c);
(E) a step of activating the egg into which the cell nucleus has been injected in step (d), and transplanting the nuclear transfer embryo after the activation treatment to the oviduct or uterus of a sow;
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