#author("2017-08-26T11:05:45+02:00","default:hisaom","hisaom")
[[トップページ]]

-140512 Gap-Repair Cloningについて、実験医学・クローズアップ実験法で紹介します。本ページの内容について、具体例を上げて紹介しています。

-101115 &color(red){GRCがinvitrogenからkit化されて発売されています。};GeneArt® Seamless Cloning & Assemblyという名前です。酵母のコンピテントセルやトランスフォーメーション試薬、大腸菌のコンピテントセル等もすべて含まれたキットです。これで、GRCも一般に広まることになるでしょう。このメージの役割も終わりかもしれません。ただし、だいたい一反応5,000円。ぼったくりです。

*Gap-Repair cloningを使おう! [#qc587cc6]
このページでは、簡便・高効率・安価なDNAクローニング法、Gap-Repair Cloningについて解説します。この手法は従来の制限酵素を用いたクローニングのややこしさをすべて解消する画期的な実験手法です。もし従来の制限酵素を用いたクローニングを行っている方でクロー(苦労)されている方は、まずはぜひ[[こちら>制限酵素クローニング?]]をご覧ください。

**はじめに [#ncda4d8c]
出芽酵母の細胞内では相同組み換えの活性が非常に高く、25bp程度の相同領域があれば相同組み換えがおきてDNA断片が連結されます(正確には「組み換え修復機構」によって連結されます)。この活性を利用して、酵母細胞内で様々なプラスミドのコンストラクトを構築することができます。これを「Gap-Repair Cloning」と呼びます。~

酵母を使ったGap-Repair Cloningは低コスト・高効率で、自由度が高く複雑な構造のプラスミドの構築も可能な、非常に便利な手法です。ただ一般の研究者にはあまり知られておらず、また酵母を用いるということで多少敷居が高いと思います。そこで酵母研究者以外の方が酵母のGap-Repair Cloningを導入するかどうかを判断される材料になればと思い、ここに解説をのせることにしました。~

#本ページでは、Gap-Repair CloningをGRCと略させていただきます。
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#contentsx()
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** GRCの特徴(メリット) [#o8267f7d]

-高効率~
90%以上の確率で組み換えがおきたプラスミドが得られます。

-低コスト~
Gateway Cloningのように(市販の)特別な酵素溶液を準備する必要がありません。

-デザインの自由度が高い。~
--クローニングに用いる制限酵素部位をあれこれ考える必要がない。~
制限酵素部位をクローニングに用いませんので、制限酵素部位の情報を細かく知る必要がありません。利用する制限酵素について頭を悩まさなくてもいいのです(これは実は非常に大きなメリットです)。
--クローニングに用いる塩基配列は何でも良い。~
Gateway Cloningでは相同組み替えに特定の配列しか使えません。GRCはどんな配列を選んでもいいのです。
--部位特異的置換や欠失などが作成できる。~
上の特徴を利用すれば、特定の塩基を置換したり欠失させたりも出来ます。
--複数の断片を結合できる。~
とにかく特異的で相同な25bp程度の相同な配列さえあれば(つくれば)、複数のDNA断片が結合できます。4断片くらいならば平気でつながります。

-長い断片を利用できる~
酵母への形質転換では、大腸菌のように長い断片が入りにくいということがありません。15kbくらいのプラスミドは平気で扱うことが出来ます。

-クローニングに用いるDNAを精製する必要がない~
PCRや制限酵素で処理したDNAをそのまま酵母に形質転換しても効率はほとんど変わりません。

-酵母の表現型で選択することができる~
これは酵母研究者だけのメリットかもしれませんが、変異株の中でGRCを用いてプラスミドをクローニングすれば、「変異株を相補するかどうか」などの指標によって組み替えプラスミドをもった酵母を選択することが可能です。

**GRCの特徴(デメリット) [#kdc3b226]
もちろんGRCにもデメリットがあります。

-酵母を使えなければならない。~
これが一般の研究者にとってもっとも大きな敷居だと思います。酵母は大腸菌が扱えればだいたい培養することは可能ですが違いもあります。ちなみに下に書きましたがYeast Two-Hybrid Systemが使えているならば、GRCでの酵母の扱いはまったく問題ありません。[[一部培養細胞を使っている研究室では、酵母は著しく嫌われているようです。:http://yeast-forum.org/YGSJwiki/index.php?ML%A4%CE%C5%EA%B9%C6%A4%AB%A4%E9#j13deffa]]そのあたりの事情も加味する必要があるかもしれません。

-時間・手間がかかる。~
酵母でクローニングするために、酵母が生えてくるまでの3日ほど余計に時間がかかります。また、プラスミドの構造を確かめるためには酵母からプラスミドDNAをリカバーして大腸菌に形質転換し直してやる必要があります。ただしクローニングに用いるDNA精製に手間がかからないことや成功率が高いことを考慮すると結局GRCの方が早いのではないかと思いますが。

-プライマーが長くなる(コスト?)。~
制限酵素でのクローニングよりは多少プライマーが長くなります。これもDNA精製のコストと相殺されるように思いますが。

-PCRによるエラー~
これを嫌う研究者はいます。しかしGRCに限らず、現在PCRを使わないコンストラクションというのはほとんど考えられない時代になりました。なるべくエラーの少ないPCR酵素を用いて、最後は塩基配列の確認を行うなどの対処が必要になるでしょう。

**GRCを行うには [#a0ff1607]
以下に、実際にGRCを行うために必要な道具立てを書きます。

-酵母を使った実験系~
酵母を研究対象としてない研究室で、GRCを行うことが出来るかどうかの一番良い基準は、その研究室で''Yeast Two-Hybrid System(Y2H)が動いているか''だと思います。酵母の株や培養系、酵母の形質転換法、酵母からのプラスミドリカバー法などは、Y2Hのものをそのまま利用できます。

-クローニングに用いる酵母用のプラスミド~
その上で''目的の断片を組み込むプラスミド''が必要になります。このプラスミドは、出芽酵母用の複製起点と選択マーカーを持っていなければなりません。下記の永野先生のシステムを使えば、自分たちの使いたいプラスミドをそのまま用いることができます。

以上が整った上でのGRCの手順です。実際の実験の流れは図をご覧ください。
#br
#ref(http://hismoriya.com/HMwiki/media/GRC1.png,wrap,center,720x540,50%)
#ref(http://tenure5.vbl.okayama-u.ac.jp/~hisaom/HMwiki/media/GRC1.png,wrap,center,720x540,50%)
#br
+組み込みたいDNA(インサート)を用意する。~
両端に、ベクターと相同な25bp以上の領域((もっと短くても良いという人もいます。これは最適化されたものではありません))をもつDNA断片を準備します。PCRで行うのがもっとも一般的かと思います((私たちはPCRを50μLの系で、正確性の高いPCR(KODplusなど)で行っています。))。
--3の形質転換で、それをそのまま(未精製で)用いる。
+組み込まれるベクターを用意する。~
ベクターを制限酵素で消化します。組み込みたい領域の内側であればどこで切断されていてもいいです((私たちは、ベクターは通常ミニプレップしたもの(50μL)を10μL用いて、50μL中で制限酵素消化したものを用いています。))。以下はTipsです。
--ベクターは、2カ所以上で切断した方がバックグラウンド(セルフライゲーション)が低い。
--ベクターは、制限酵素で1日以上消化したものの方がバックグラウンドが低い(4℃保存)。
--熱処理や、アルカリフォスファターゼ処理は不要。
+1と2を混ぜて酵母を形質転換し、プラスミド用の選択マーカー(一般的には栄養要求性)で形質転換体を選択する((私たちは、通常約300μLの形質転換溶液の中に50μLの(1)と2μLの(2)を混ぜて形質転換しています。))。このときベクターのみをコントロールとして形質転換しておけばどれくらいの効率でインサートが入るのかを評価することが出来ます。
+生えてきたコロニーを培養し、プラスミドをリカバー、大腸菌に形質転換し直してプラスミドの構造を確認する。

**GRCの活用事例 [#w2b94e1f]
GRCは様々に応用できます。例えば以下のように用いれば、部位特異的変異を非常に簡単に導入できます。

#ref(http://hismoriya.com/HMwiki/media/GRC3.png,wrap,center,720x540,50%)
#ref(http://tenure5.vbl.okayama-u.ac.jp/~hisaom/HMwiki/media/GRC3.png,wrap,center,720x540,50%)

その他、以下のような用途があります。
-GFPなどとの融合遺伝子の作成
-ノックアウトやトランスジェニックのためのコンストラクトの作成
-新しいベクターの構築

**GRCのその他の解説と関連情報[#tb3f5d1f]
-[[佐賀大学・永野幸生先生のページ:http://www.iac.saga-u.ac.jp/lifescience/researches/research2.htm]]~
「GRCをやりたいが、酵母用のベクターを持っていない」という方に朗報です。永野先生らの「ヘルパープラスミド」を用いれば、酵母用のベクターでなくてもインサートと酵母用の複製起点とマーカーを同時にGRCすることが出来ます。リクエストすれば分与もしていただけるようです。さらにこのページはGRCについて詳細に説明されていますので大変参考になると思います。~

-&ref_paper(2828185);~
酵母でのGap-Repair Cloningの(おそらく)始めての報告となる論文。この手法によって様々なプラスミドを構築している。

-&ref_paper(20488990);~
2014.5.13追記:合成DNAからマイコプラズマの全ゲノムDNAを再構築した研究でも、酵母の相同組換えが使われている。

**最後に [#j3a54fad]
GRCは非常に自由度の高いDNA構築系です。従来の制限酵素ーライゲーションの系や、Gateway Cloningのような、制限酵素部位や組み換え配列の制約を一切受けません。したがって、非常に自由に思い通りのデザインでDNAを操作することが出来ます。「バカでもできる」と書きましたが、これはつまり作成したいコンストラクトのデザインに上記の制約のせいで「頭を悩ませる必要がない」ということを意味します。GRCを活用して「あっと驚くコンストラクト」を作成してみませんか?

**FAQ [#w91912e8]
「最後に」の後ですが、GRCについていくつか疑問にあがりそうなことを書いてみました。その他ご意見ご質問等ありましたら、下記コメント欄にお書きください。

-酵母を扱ったことがないのだが?~
「バイオ実験イラストレイテッド〈7〉使おう酵母できるTwo Hybrid」という書籍をお薦めします。

-なぜGRCは一般に広まっていないのか?~
一つには、酵母を使わなければならない(手間がかかる、一部の研究者から嫌われている(上述))ということがあると思います。しかし、もう一つ大きな理由として''試薬業者にとってこれを宣伝するメリットがない''ということが挙げられると思います。 Gateway Cloningのように酵素と抱き合わせで売れば、売っただけInvtrogenは儲かるのです。もっと便利で安価な方法があるのに・・・私がこのページを作った理由です。
--101115 &color(red){GRCがinvitrogenからkit化されて発売されました};(私たちは関係ありません)。GeneArt® Seamless Cloning & Assemblyという名前です。酵母のコンピテントセルやトランスフォーメーション試薬、大腸菌のコンピテントセル等もすべて含まれたキットです。これで、GRCも一般に広まることになるでしょう。ただし、だいたい一反応5,000円。ぼったくりです。

-他の生物でGRCは出来ないのか?~
実は大腸菌で出来るという報告があります。((例えば、http://proteome.wayne.edu/RecombCloning2.pdf )) 
これでゲノムワイドコレクションを作っていると主張しているグループもあります。もし大腸菌で出来れば非常に便利なので、私たちも試したことがあります。実は一度成功しました。しかし全くうまく行かないときもあったりして系が安定していないので今は使っていません。~
分裂酵母でも出来るという報告があります((例えば、&ref_paper(15157886);))。%%私たちも試したことがありますがうまく行きませんでした。今のところ出芽酵母が一番安定したGRCのホストだと思っています。%%~
私たちは最近、分裂酵母でのGRCが高効率で成功するということを確認しました。これについて、[[PLoS Oneに2010年3月に発表しました:http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0009652]]。私たちの発表とほぼ同時(正確には先方が3日前)に、島根大学の川向先生のグループも分裂酵母のでのGRCの効率について、[[Bioscience, Biotechnology, and Biochemistry誌に発表されています:http://www.jstage.jst.go.jp/article/bbb/advpub/0/advpub_1002181861/_article/-char/ja/]]。~
私たちと川向先生のグループではGRCの効率が違う(>70%と~20%)ので、今後汎用化のためにはこの違いをはっきりとさせる必要があるようにおもいます。

-&color(red){Clontechのin-Fusionとの違いは?};~
最近、[[Clontech(TAKARA)からin-Fusionという試薬が売り出されています:http://catalog.takara-bio.co.jp/clontech/product/list.asp?catcd=B1000588&subcatcd=B1000606#B1000606]]。これは相同組み替え酵素を利用して、in vitroでGCRとほぼ同じことを行なうものです。試薬は定価ベースで一回あたり1700円から2000円ですが、「酵母を使わなくてよい」というメリットは大きいように思います。カタログを見る限りでは、GCRにはない制約がいくつかあるようですが、企業が売り出すといろいろなテストケースが出て来て改良されてくるでしょうから(トラブルシューティングも充実するでしょうし)、GCRの最大のライバルになるように思います。

-GRCに不可能はないか?~
いろいろあると思います。最大の弱点は、リピートが作れないことです。相同な配列があるとそこで組み換えを起こしてしまうからです。
いろいろあると思います。%%最大の弱点は、リピートが作れないことです。%%相同な配列があるとそこで組み換えを起こしてしまうからです。~
2012.12.19追記:最近リピートも作れる方法を考案しました。興味がある方にはこっそりお教えします。

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