私たちは感謝シンガポール（ETRP 1201102）の国家環境庁からの資金援助を認める、シンガポール国立研究財団（NRF-CRP5-2009-03）、シンガポールの科学技術研究庁の競争研究プログラム（ 1324004108）、グローバルR＆Dプロジェクトの計画、知識経済部、大韓民国（N0000677）、国防脅威削減局（DTRA、HDTRA1-13-1-0037）と合成生物学シンガポール国立大学のイニシアティブ（ DPRT / 943/9月14日）。

Yの1世代KU70欠失株リポリティカ <ol><li>破壊カセットの構築 注：ポリメラーゼ連鎖反応（PCR）増幅に使用されるすべてのプライマーは、 表1を参照のこと。 <ol><li> LEU2発現カセットをPCR増幅するために設計プライマー20は、Yから（ 表1参照します） リポリティカ（lipolytica）発現ベクターおよび長いフォワードプライマー（＃1、 表1）を用いてLEU2カセットの5 &#39;および3&#39;末端にLoxP部位を導入し、それぞれ、長逆方向プライマー（ 表1＃2）。クローニングプロセスの後続のステップのために追加の制限部位を導入するために、プライマー＃1におけるBamHI制限部位を加えます。 </li><li> 表2に詳述するように、高忠実度DNAポリメラーゼを用いてPCRを行います。 </li><li> PCR purificatioを使用して、PCR産物のLoxP-プロモーター-LEU2-ターミネーターのLoxPカセットを精製製造元の指示に従って、n個のキット。製造業者の説明書21に記載の精製されたPCR産物に3&#39;-Aオーバーハングを加えます。 表3の通りプラスミドT-LEU2を得るためのTAクローニングベクターにAテールPCR産物を連結するために使用T4 DNAリガーゼ。 </li><li> PCRは、Yからプライマー＃3 /＃4を使用してKU70遺伝子の1キロバイトの5 &#39;上流配列を増幅しますリポリティカ Po1g 表2に従って、ゲノムDNA 22は、製造業者の説明書に従ってPCR精製キットを用いてPCR産物を精製します。 <ol><li>ダブル。 表4のとおりのSacIIおよびBam HI酵素で精製したPCR産物とT-LEU2プラスミドの両方を消化し ​​、製造元の説明書に従ってPCR精製キットを用いて、消化混合物を精製します。精製を連結するためにT4 DNAリガーゼを使用して、 サック IIにPCR産物を消化/ T-LEU2ののBamHI部位を生成しますプラスミドT-LEU2-5E 表3のとおり。 </li></ol></li><li> PCRは、Yからのプライマーを＃5 /＃6を使用してKU70遺伝子の1キロバイトの3 &#39;下流配列を増幅しますリポリティカ Po1g 表2に従って、ゲノムDNA 22は、製造業者の説明書に従ってPCR精製キットを用いてPCR産物を精製します。ダブル精製したPCR産物と表4のとおりのNot Iおよびんで酵素とT-LEU2-5Eプラスミドの両方を消化。 <ol><li>製造業者の説明書に従ってPCR精製キットを用いて、消化混合物を精製します。その後、精製を結紮およびNot I / んで 表3に従ってT4 DNAリガーゼを用いてプラスミドT-KOを生成するT-LEU2-5EのI部位にPCR産物を消化し ​​ました。 </li></ol></li><li>破壊カセットを生成するために、表4のとおりのSacIIおよびんで I酵素によるプラスミドのT-KOダイジェスト（ 図1 </stronグラム&gt;）。製造業者の説明書に従ってPCR精製キットを用いて消化したDNA断片を精製します。 </li></ol></li><li>コンピテントセルの調製およびYの変換Po1g株リポリティカ <ol><li>コンピテント細胞調製<ol><li> Yのコロニーを植菌YPD培地10ml（1％酵母エキス、2％ペプトン、2％デキストロースおよび50mMのクエン酸緩衝液pH 4.0）を100mlフラスコ中で、新鮮な酵母抽出物-ペプトン-デキストロース（YPD）プレートからリポリティカ Po1g株。飽和するまで20時間（分光光度計を用いて測定し、OD約15の600）を225 rpmで30℃の振盪インキュベーター中でインキュベートします。 </li><li>室温で5000×gで5分間遠心して細胞をペレット化。 20ミリリットルのTris-EDTA（TE）緩衝液（10mMのTris、1mMのEDTA、pH7.5）で細胞を洗浄し、ステップ1.2.1.2で説明したように、細胞をペレット。 （酢酸で調節してpH 6.0）を1mlの0.1M酢酸リチウム中で細胞を再懸濁し、そして室テンペで10分間インキュベートrature。 </li><li>滅菌1.5ミリリットルチューブにテント細胞（100μl）を等分。直下変換ステップに進む、または長期保存のために-80℃で、最終濃度25％（v / v）を、店舗にグリセロールを加えます。 </li></ol></li><li>変換<ol><li>穏やかにコンピテント細胞100μlと共に変性サケ精子DNA（10 mg / mlで）、精製破壊カセット1-5μgの10μLを混合し、15分間30℃でインキュベートします。 </li><li> （0.1 M酢酸リチウム液pH 6.0に溶解した）40％ポリエチレングリコール（PEG）-4000の700μl加え、よく混ぜ、60分間225rpmで30℃の振盪インキュベーター中でインキュベートします。 注：（典型的には、従来の酵母の形質転換に使用される）の代わりにPEG-3350の、4000の平均分子量を有するPEGを使用することが重要です。 </li><li>熱は、60分間39℃の水浴中に管を配置することによって、形質転換混合物に衝撃を与えます。 </li><li> 1ミリリットルをYPD培地を追加し、30°Cおよび225 rpmで2時間回復。室温で1分間、9000×gで遠心し、上清を除去し、TE緩衝液1mlでペレットを再懸濁します。 </li><li>室温で1分間9000×gの遠心分離により細胞をRepelletし、上清を捨てます。 100μlのTE緩衝液にペレットを再懸濁し、選択プレート（ 例えば 、ロイシン欠乏プレート）上にプレートし、2〜3日間30℃でインキュベートします。 </li><li> 4〜10の単一コロニーを選択し、YPD培地2mlに別々に接種します。 225 rpmで30℃の振盪インキュベーター中で一晩インキュベートします。それぞれのプライマー＃55 /＃56、及び＃56 /＃57、の2セットを用いて、 表5のとおり形質転換体22からのゲノムDNAのPCR分析によって陽性コロニーを識別します。 注：私は、直鎖状にpYLEX1ベクターがY.に変換されるわけではありません数をカウントすることにより形質転換効率を決定するためにPo1gテント細胞リポリティカμgのプラスミド当たりのコロニー形成単位（CFU）のDNAは、23を用います。 </li></ol></li></ol></li></ol> 2.マーカーレスキュー<ol><li> Cre発現プラスミドの構築<ol><li> pYLEX1-CREとpYLEX1-HPHの構築<ol><li>デザインプライマー20は、CreおよびHPHのオープンリーディングフレームをPCR増幅します。フォワードプライマー＃82と＃84に開始コドンの上流の余分なアデニンヌクレオチドを追加し、PCRは、CREのオープンリーディングフレームを増幅するリバースプライマー＃83と＃85の中に終止コドンの下流のKpnI制限部位を挿入し、 表2のとおりpSH69（アクセッション番号HQ412578）24からHPH。 </li><li> CRE、製造業者の説明書に従ってPCR精製キットを使用して、HPH断片の両方を精製します。 TABL通りのKpn I酵素で精製CREおよびHPH断片の両方を消化E 4。Pmlに基づくおよびKpnIでpYLEX1プラスミドは表4の通り酵素消化倍増。当たりの製造者の指示通りPCR精製キットを用いて、消化混合物を精製します。 </li><li> Y.のPmlに基づく I / KpnIのサイトに精製し、消化CREおよびHPH断片を連結するには、 表3の通り、T4 DNAリガーゼを使用します発現ベクターリポリティカ 、それぞれ、pYLEX1-CREとpYLEX1-HPHを得ました。 </li></ol></li><li> pSL16-CRE-HPHの構築<ol><li> PCRは、プライマーに＃86を使用してpYLEX1-CREからのcreのプロモーター-遺伝子-ターミネーターカセットを増幅/＃87、表2のとおりのSal I / PstIの制限部位に隣接する。メーカーの指示に従って、PCR精製キットを用いてPCR産物を精製。 <ol><li>精製したPCR産物およびSal IでpSL16-CEN1-1（227）プラスミド25の両方をダブル消化し ​​、PST 表4に従って、I酵素は、製造業者の指示に従って、PCR精製キットを用いて、消化混合物を精製します。そして、 表3に従ってT4 DNAリガーゼを用いpSL16-CREを作成するために対応する部位にpSL16-CEN1-1（227）に精製し、消化し ​​たPCR産物を連結。 </li></ol></li><li> PCRは、＃88 /＃89は、 表2の通りのXho I / のBglII制限部位に隣接するプライマーを用いてpYLEX1-HPHからHPHのプロモーター-遺伝子-ターミネーターカセットを増幅する。製造業者の説明書に従ってPCR精製キットを用いてPCR産物を精製します。 <ol><li>ダブル。精製されたPCR産物および表4のとおりのXho IおよびBgl II酵素とpSL16-CREプラスミドの両方を消化し ​​、製造元の説明書に従ってPCR精製キットを用いて、消化混合物を精製します。次に、対応するでpSL16-CREに精製し、消化したPCR産物を連結サイトは、 表3に従ってT4 DNAリガーゼを用いpSL16-CRE-HPHを生成します。 注：結果のCre発現プラスミド、pSL16-CRE-HPHは、選択可能なハイグロマイシンBのマーカーを保有し、セントロメアおよびエピソーム的に複製するベクター（ 図2）です。 </li></ol></li><li> 表4の通り（ベクターからの挿入を切り出すために、 例えば 、 サル I / PstIの ）適切な制限酵素で消化することにより全ての構築物を確認してください。 </li></ol></li></ol></li><li> Creリコンビナーゼ媒介マーカーレスキュー<ol><li>上記のセクション1.2に記載の形質転換プロトコルに従ってKU70ノックアウト株のコンピテント細胞にpSL16-CRE-HPHを変換します。プレートは、YPDプラスハイグロマイシンB上に細胞（YPDH）プレート（400 / mlのハイグロマイシンBを含む）に形質転換し、2〜3日間30℃でインキュベートします。 </li><li>正識別するためのプライマー＃84 /＃85を用いて、 表5の通りコロニーPCRを行います変換後のpSL16-CRE-HPHプラスミドを含むコロニー。 注：フォワードプライマー＃84およびリバースプライマー＃85は、hph遺伝子（ 表1）のコード領域の内側に配置されています。唯一の陽性クローンをPCR反応に特異的なPCR産物を生成します。 </li><li>陽性クローンを選択し、YPDH培地2mlに接種し、飽和するまで一晩225rpmで30℃の振盪インキュベーター中でインキュベートします。分光光度計を用いて細胞のOD 600を測定します。 〜15のOD 600で細胞を回収します。室温で5分間、5000×gで遠心分離し、滅菌水2mlで1回洗浄。 </li><li>初期OD 0.1の600でYPD培地2mlに細胞を再接種し、細胞が一晩225rpmで30℃の振盪インキュベーター中で成長することができます。単一コロニー23を分離するYPDプレート上にストリーク一晩細胞培養物。 YPD、YPDH、およびロイシン欠乏プレート23上にレプリカプレートコロニー</s&gt;アップ。 注：両方のLEU2マーカーを失っているとpSL16-CRE-HPHプラスミドのみをYPDプレート（ 図3）上に成長させることができるコロニーを。 </li></ol></li></ol>アルコール脱水素酵素およびアルコールオキシダーゼ遺伝子の3削除<ol><li> Y.で検索を実行します削除の26の遺伝子候補を同定するために基本的なローカル配列検索ツール（BLAST）を使用してリポリティカゲノムデータベース。入力Sのタンパク質配列BLAST検索ツールにセレビシエのアルコールデヒドロゲナーゼIを（http://www.genome.jp/tools-bin/search_sequence?prog=blast&amp;db=yli&amp;seqid=aa）。 注：BLAST検索ツールは、Yに最も類似したタンパク質配列を返しますリポリティカゲノムデータベース。 </li><li> ＃56へのプライマー＃7（ 表1）を用いたPCRによって削除カセットを構築し、上記のセクション1.1に記載したのと同じ方法を用いて、制限酵素消化およびライゲーション反応を行います。 </li><li>予備校KU70ノックアウト株のコンピテントセルは、ある変換を行い、上記のセクション1.2に記載のプロトコルに従うことにより、＃81（ 表1）に＃55のプライマー、＃58で陽性コロニーを同定するためのPCRを実施します。 注：例として、YALI0E17787g遺伝子欠失のための手順は、 図4及び5に記載されています。相同組換え率に1キロバイトおよび2キロバイト長い相同領域の効果が報告されています。 </li></ol>

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Microbiol. 49 (3), 469-472 (2011).</li><li>Matsuoka, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+regions+essential+for+the+function+of+chromosomal+replicator+sequences+from+Yarrowia+lipolytica.">Analysis of regions essential for the function of chromosomal replicator sequences from Yarrowia lipolytica.</a> Mol. Gen. Genet. 237 (3), 327-333 (1993).</li><li>Kretzschmar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+homologous+integration+frequency+in+Yarrowia+lipolytica+strains+defective+in+non-homologous+end-joining.">Increased homologous integration frequency in Yarrowia lipolytica strains defective in non-homologous end-joining.</a> Curr. Genet. 59 (1-2), 63-72 (2013).</li><li>Lorenz, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerase+Chain+Reaction:+Basic+Protocol+Plus+Troubleshooting+and+Optimization+Strategies.">Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies.</a> J. Vis. Exp. (63), e3998 (2012).</li><li>Kobs, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+blunt-end+DNA+fragments+into+the+pGEM-T+Vector+Systems.">Cloning blunt-end DNA fragments into the pGEM-T Vector Systems.</a> Promega Notes. 62, 15-18 (1997).</li><li>Sanders, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aseptic+laboratory+techniques:+plating+methods.">Aseptic laboratory techniques: plating methods.</a> J. Vis. Exp. 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Bioeng. 105 (6), 571-578 (2008).</li><li>McGinnis, S., Madden, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BLAST:+at+the+core+of+a+powerful+and+diverse+set+of+sequence+analysis+tools.">BLAST: at the core of a powerful and diverse set of sequence analysis tools.</a> Nucleic Acids Res. 32 ((Web Server issue)), W20-W25 (2004).</li><li>Madzak, C., Tr&#233;ton, B., Blanchin-Roland, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strong+hybrid+promoters+and+integrative+expression/secretion+vectors+for+quasi-constitutive+expression+of+heterologous+proteins+in+the+yeast+Yarrowia+lipolytica.">Strong hybrid promoters and integrative expression/secretion vectors for quasi-constitutive expression of heterologous proteins in the yeast Yarrowia lipolytica.</a> J. Mol. Microbiol. 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The authors have nothing to disclose.

この研究のための私たちの目標は、Yに標的遺伝子ノックアウトの迅速かつ効率的な生成を可能にすることですPo1g株リポリティカ 。いくつかの考慮事項は、これを達成するために対処する必要があります。まず、高変換効率が必要です。したがって、Y.ための効率的で便利な化学的形質転換プロトコルリポリティカ Po1g株を本研究に記載されました。 PEG-4000を...

サッカロマイセス・セレビシエ 、 ヤロウィアリポリティカ、型にはまらない酵母とは異なり、環境条件1,2の変化に応じて、酵母または菌糸体の形で成長することができます。したがって、この二形酵母は真菌の分化、形態形成および分類3,4,5の研究のための良いモデルとして使用することができます。一般に広く、有機酸、ポリアルコール、芳香化合物、乳化剤及び界面活性剤-6,7,8,9-などの食品添加物の多様を生成するために使用される安全な（GRAS）酵母種とし ​​て見なされます。これは絶対好気性自然細胞乾燥重量10の70％まで、多量、 すなわちに脂質を蓄積することができる周知の油性酵母です。また、栄養素11,12,13として廃資源における残基の異なる種類を含む、成長のための炭素源の広いスペクトルを利用することができます。これらのユニークな機能のすべてがYを作ります以下のための非常に魅力的リポリティカ様々なバイオテクノロジー用途。 Yの全ゲノム配列が、 リポリティカが 14,15を公開されている、この型破りな酵母の遺伝子操作は、他の酵母種よりも複雑です。まず、この酵母種の変換は、それほど効率的により安定的かつ効率的な形質転換系16,17が存在しないことです。全く自然なエピソームプラスミドシステムは、この酵母18で発見されていないように、第2の線形発現カセットの面倒なゲノム組み込みは、一般に、目的の遺伝子の発現のために使用されます。この酵母で正確な相同組換えを介して効率遺伝子ターゲッティングが低く、ほとんどの統合イベントは非相同末端結合（NHEJ）19を介して発生するため、遺伝的ノックアウトおよびノックインの第三に、世代が制限されています。 本研究では、Yのための最適化された形質転換プロトコルを報告しますリポリティカ </em&gt;の、簡単迅速、効率的かつ再現性のあるPo1g株。正確な相同組換えの頻度を高めるために、我々は、NHEJ経路における重要な酵素をコードするKU70遺伝子を削除しました。最適化された形質転換プロトコルを用いて、1キロバイト、Y.のKU70遺伝子のフランキング相同領域を含む直鎖ノックアウトカセットを形質転換することによってリポリティカ Po1gは正常に削除されました。この遺伝子の欠失方法のロバスト性は、その後Po1g KU70Δ株において、アルコールデヒドロゲナーゼ及びアルコールオキシダーゼ遺伝子を標的にすることによって実証されました。これは、KU70欠損株は、野生型Po1g株よりも標的相同組換えを介した遺伝子のかなり高い効率を示すことが観察されました。また、ハイグロマイシンB耐性マーカーを保有する複製Cre発現プラスミドは、マーカーレスキューを実行するように構築しました。マーカーレスキューは番目に遺伝子標的の複数のラウンドを容易にEは、遺伝子欠失突然変異体を得ました。遺伝子欠失に加えて、ここに記載の遺伝的形質転換および遺伝子欠失のための我々のプロトコルは、特定の遺伝子座に遺伝子を挿入するために適用することができ、およびY.に部位特異的変異を導入することリポリティカゲノム 。

<table border="0" cellpadding="0" cellspacing="0" width="605">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:140px;">Reagent/Material</td>
			<td colspan="3" style="width:465px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Oligonucleotide primers</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>25 nmole&#160; DNA oligos</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Y. lipolytica strain Po1g</td>
			<td>Yeastern Biotech</td>
			<td></td>
			<td style="width:207px;">leucine auxotrophic derivative of the wild-type strain W29 (ATCC 20460)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vector pYLEX1</td>
			<td>Yeastern Biotech</td>
			<td>FYY203-5MG</td>
			<td>Y. lipolytica expression vector&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">E. coli TOP10&#160;</td>
			<td>Invitrogen</td>
			<td></td>
			<td>For cloning and propagation of plasmids</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pGEM-T vector&#160;</td>
			<td>Promega</td>
			<td>A3600</td>
			<td>TA cloning vector</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QIAprep Spin Miniprep Kit</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td>For plasmid isolation</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:140px;">Wizard SV Gel and 
			PCR Clean-Up System</td>
			<td>Promega</td>
			<td>A9282</td>
			<td style="width:207px;">Extract DNA fragments from agarose gels 
			and purify PCR products from an amplification reaction</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:140px;">The iProof high-fidelity 
			DNA polymerase</td>
			<td>Bio-Rad</td>
			<td>172-5302</td>
			<td>High-fidelity DNA polymerase</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BamHI&#160;</td>
			<td>New England Biolabs</td>
			<td>R0136S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BglII&#160;</td>
			<td>New England Biolabs</td>
			<td>R0144L</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KpnI</td>
			<td>New England Biolabs</td>
			<td>R0142S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NdeI&#160;</td>
			<td>New England Biolabs</td>
			<td>R0111S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NotI</td>
			<td>New England Biolabs</td>
			<td>R0189L</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PmlI</td>
			<td>New England Biolabs</td>
			<td>R0532S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PstI&#160;</td>
			<td>New England Biolabs</td>
			<td>R0140S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SacII</td>
			<td>New England Biolabs</td>
			<td>R0157S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SalI</td>
			<td>New England Biolabs</td>
			<td>R0138S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">XhoI</td>
			<td>New England Biolabs</td>
			<td>R0146L</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">T4 DNA ligase</td>
			<td>New England Biolabs</td>
			<td>M0202L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Taq DNA polymerase&#160;</td>
			<td>Bio-Rad</td>
			<td>M0267L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ampicillin</td>
			<td>Gibco-Life Technologies</td>
			<td>11593-027</td>
			<td>Antibiotics</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hygromycin B</td>
			<td>PAA</td>
			<td>P21-014</td>
			<td>Antibiotics</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GeneRuler 1 kb DNA ladder</td>
			<td>Thermo Scientific</td>
			<td>SM0312</td>
			<td>1 kb DNA ladder</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PEG4000</td>
			<td>Sigma</td>
			<td>95904-F</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris</td>
			<td>Promega</td>
			<td>H5135</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Bio-Rad</td>
			<td>161-0729</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Salmon Sperm DNA</td>
			<td>Invitrogen</td>
			<td>15-632-011</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lithium Acetate</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetic acid</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass beads (425-600 &#181;m)</td>
			<td>Sigma</td>
			<td>G8772</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNAse A</td>
			<td>Thermo Scientific</td>
			<td>EN0531</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNA Loading Dye</td>
			<td>Thermo Scientific</td>
			<td>R0611</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bacto Yeast Extract</td>
			<td>BD</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bacto Peptone</td>
			<td>BD</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">D-Glucose</td>
			<td>1st Base</td>
			<td>BIO-1101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">YNB without amino acids</td>
			<td>Sigma</td>
			<td>Y0626</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DO Supplement-Leu</td>
			<td>Clontech</td>
			<td>630414</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycerol</td>
			<td>Sigma</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Difco LB Broth</td>
			<td>BD</td>
			<td>244620</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Difco LB Agar</td>
			<td>BD</td>
			<td>244520</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bacto Agar</td>
			<td>BD</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PCR machine</td>
			<td>Biorad</td>
			<td>T100 Thermal Cycler</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Water bath</td>
			<td>Memmert</td>
			<td>WNB 14</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stationary/Shaking Incubator</td>
			<td>Yihder</td>
			<td>LM-570RD</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thermo-shaker</td>
			<td>Allsheng</td>
			<td>MS-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micro centrifuge</td>
			<td>Eppendorf</td>
			<td>5424R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Spectrophotometer</td>
			<td>Eppendorf&#160;</td>
			<td>BioPhotometer plus</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gel imager</td>
			<td>GE</td>
			<td colspan="2">Amersham Imager 600</td>
		</tr>
	</tbody>
</table>

genetic engineering of an unconventional yeast for renewable biofuel and biochemical production

ヤロウィアリポリティカは 、非病原性の二形性と厳密に好気性の酵母種です。その独特の生理機能や代謝の特性により、この型破りな酵母は真菌の分化の基本的な性質の研究のための良好なモデルだけではありませんが、また、生化学的生産と大規模な遺伝子操作を必要とする様々な生物工学的適用のための有望な微生物のプラットフォームです。 Y.のが、遺伝子操作リポリティカは 、主KU70遺伝子に起因することができ、効率的かつ安定的な遺伝的形質転換システムの欠如ならびに非相同組換えの非常に高い割合に限定されています。ここでは、効率的な遺伝的形質転換のためにとYにおける遺伝子欠失のための簡単かつ迅速なプロトコルを報告しますリポリティカ Po1g。 Y.への外因性DNAの効率的な形質転換のためにまず、プロトコルリポリティカ Po1gを確立しました。セコndは、標的遺伝子のさらなる削除のために強化された二重交差相同組換え率を達成するために、KU70遺伝子は、1kbの相同性ア ​​ームを担持破壊カセットを形質転換することにより削除されました。第三に、KU70遺伝子の欠失後の増強遺伝子欠失の効果を実証するために、我々は、個別にKU70ノックアウトプラットフォーム株に同じ手順を使用して、アルコールデヒドロゲナーゼおよびアルコールオキシダーゼをコードする11標的遺伝子を削除しました。これは、正確な相同組換えの割合が削除のために0.5％未満の実質的に増加することが観察されました Po1g KU70Δで11標的遺伝子の単一遺伝子欠失のための33％-71％にPo1gでKU70遺伝子の。ハイグロマイシンB耐性マーカーを有する複製プラスミドとのCre / loxPシステムを構築し、酵母ノックアウト株における選択マーカー遺伝子は、最終的にタージェ複数回のを容易にするために、Creリコンビナーゼの発現により除去しましたテッド遺伝子操作。得られた単一遺伝子欠失変異体は、バイオ燃料および生化学的生産において潜在的な用途を有します。

線形化Y.リポリティカ（lipolytica）発現ベクターは、Yのゲノム中のpBRドッキングプラットフォームに挿入し単一交差組換え27を行うことにより、 リポリティカ Po1g株。本研究で確立された迅速な化学変換の手順を使用して、線形化Y.リポリティカ（lipolytica）発現ベクターは、正常&gt; 100 CFU /μgのDNAの形質転換効率で野生型Po1g株...

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Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production

We herein report methods on the molecular genetic manipulation of the Yarrowia lipolytica Po1g strain for improved gene deletion efficiency. The resulting engineered Y. lipolytica strains have potential applications in biofuel and biochemical production.

再生可能バイオ燃料および生化学的生産のための独創的な酵母の遺伝子工学

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic ...

genetic-engineering-an-unconventional-yeast-for-renewable-biofuel

Research

JoVE Journal

Genetics

13.5K ビュー.  National University of Singapore. We herein report methods on the molecular genetic manipulation of the Yarrowia lipolytica Po1g strain for improved gene deletion efficiency. The resulting engineered Y. lipolytica strains have potential applications in biofuel and biochemical production.

ビデオ: Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

ハイスループットプロトプラスト単離と変革のためのロボットプラットフォーム

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

遺伝学

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 nuclease or a nuclease-dead dCas9 fusion protein) is targeted to a specific site in the genome by a small synthetic RNA known as the guide RNA, or gRNA. The bipartite nature of the CRISPR system enables its use in screening approaches since plasmid libraries containing expression cassettes of thousands of individual gRNAs can be used to interrogate many different sites in a single experiment. 
To date, gRNA sequences for the construction of libraries have been almost exclusively generated by oligonucleotide synthesis, which limits the achievable complexity of sequences in the library and is relatively cost-intensive. Here, a detailed protocol for CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a simple and cost-effective method for the generation of highly complex gRNA libraries based on enzymatic digestion of input DNA, is described. Since CORALINA libraries can be generated from any source of DNA, plenty of options for customization exist, enabling a large variety of CRISPR-based screens.

Methods for generating large-scale gRNA libraries should be simple, efficient and cost-effective. We describe a protocol for the production of gRNA libraries based on enzymatic digestion of target DNA. This method, CORALINA (comprehensive gRNA library generation through controlled nuclease activity) presents an alternative to costly custom oligonucleotide synthesis.

大規模な gRNA ライブラリ生産 DNA の任意のソースからの普遍的なプロトコル

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 ...

Production and Purification of Baculovirus for Gene Therapy Application

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a promising vector for gene therapy application. Here, baculovirus is produced by transient transfection of the baculovirus plasmid DNA (bacmid) in an adherent culture of Sf9 cells. Baculovirus is subsequently expanded in Sf9 cells in a serum-free suspension culture until the desired volume is obtained. It is then purified from the culture supernatant using heparin affinity chromatography. Virus supernatant is loaded onto the heparin column which binds baculovirus particles in the supernatant due to the affinity of heparin for baculovirus envelop glycoprotein. The column is washed with a buffer to remove contaminants and baculovirus is eluted from the column with a high-salt buffer. The eluate is diluted to an isotonic salt concentration and baculovirus particles are further concentrated using ultracentrifugation. Using this method, baculovirus can be concentrated up to 500-fold with a 25% recovery of infectious particles. Although the protocol described here demonstrates the production and purification of the baculovirus from cultures up to 1 L, the method can be scaled-up in a closed-system suspension culture to produce a clinical-grade vector for gene therapy application.

In this protocol, baculovirus is produced by transient transfection of baculovirus plasmid into Sf9 cells and amplified in a serum-free suspension culture. The supernatant is purified by heparin affinity chromatography and further concentrated by ultracentrifugation. This protocol is useful for the production and purification of baculovirus for gene therapy application.

生産およびバキュロ ウイルスの遺伝子治療応用のための浄化

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a ...

High-Resolution Comparison of Bacterial Conjugation Frequencies

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons of conjugation frequency under different conditions are required to understand how the conjugative element spreads in nature. However, conventional methods for comparing conjugation frequency are not appropriate for in-depth comparisons because of the high background caused by the occurrence of additional conjugation events on the selective plate. We successfully reduced the background by introducing a most probable number (MPN) method and a higher concentration of antibiotics to prevent further conjugation in selective liquid medium. In addition, we developed a protocol for estimating the probability of how often donor cells initiate conjugation by sorting single donor cells into recipient pools by fluorescence-activated cell sorting (FACS). Using two plasmids, pBP136 and pCAR1, the differences in conjugation frequency in Pseudomonas putida cells could be detected in liquid medium at different stirring rates. The frequencies of conjugation initiation were higher for pBP136 than for pCAR1. Using these results, we can better understand the conjugation features in these two plasmids.

With an aim to understand the behaviors of various bacterial conjugative DNA elements under different conditions, we describe a protocol for detecting differences in conjugation frequency, with high resolution, to estimate how efficiently the donor bacterium initiates conjugation.

細菌の活用頻度の高解像度の比較

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons ...

Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such as initial massive cell death and low therapeutic effects, impaired their broad clinical translation. Genetic engineering of stem cells prior to transplantation emerged as a promising method to optimize therapeutic stem cell effects. However, safe and efficient gene delivery systems are still lacking. Therefore, the development of suitable methods may provide an approach to resolve current challenges in stem cell-based therapies.
The present protocol describes the extraction and characterization of human dental follicle stem cells (hDFSCs) as well as their non-viral genetic modification. The postnatal dental follicle unveiled as a promising and easily accessible source for harvesting adult multipotent stem cells possessing high proliferation potential. The described isolation procedure presents a simple and reliable method to harvest hDFSCs from impacted wisdom teeth. Also this protocol comprises methods to define stem cell characteristics of isolated cells. For genetic engineering of hDFSCs, an optimized cationic lipid-based transfection strategy is presented enabling highly efficient microRNA introduction without causing cytotoxic effects. MicroRNAs are suitable candidates for transient cell manipulation, as these small translational regulators control the fate and behavior of stem cells without the hazard of stable genome integration. Thus, this protocol represents a safe and efficient procedure for engineering of hDFSCs that may become important for optimizing their therapeutic efficacy.

This protocol describes the transient genetic engineering of dental stem cells extracted from the human dental follicle. The applied non-viral modification strategy may become a basis for the improvement of therapeutic stem cell products.

分離の特性と人間の歯小嚢細胞のマイクロ Rna を用いた遺伝子組み換え

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such ...

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other important cellular biology questions. The technologies available to genetically manipulate Dictyostelium cells are well-developed. Transfections can be performed using different selectable markers and marker re-cycling, including homologous recombination and insertional mutagenesis. This is supported by a well-annotated genome. However, these approaches are optimized for axenic cell lines growing in liquid cultures and are difficult to apply to non-axenic wild-type cells, which feed only on bacteria. The mutations that are present in axenic strains disturb Ras signaling, causing excessive macropinocytosis required for feeding, and impair cell migration, which confounds the interpretation of signal transduction and chemotaxis experiments in those strains. Earlier attempts to genetically manipulate non-axenic cells have lacked efficiency and required complex experimental procedures. We have developed a simple transfection protocol that, for the first time, overcomes these limitations. Those series of large improvements to Dictyostelium molecular genetics allow wild-type cells to be manipulated as easily as standard laboratory strains. In addition to the advantages for studying uncorrupted signaling and motility processes, mutants that disrupt macropinocytosis-based growth can now be readily isolated. Furthermore, the entire transfection workflow is greatly accelerated, with recombinant cells that can be generated in days rather than weeks. Another advantage is that molecular genetics can further be performed with freshly isolated wild-type Dictyostelium samples from the environment. This can help to extend the scope of approaches used in these research areas.

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is a popular model organism to study complex cellular processes such as cell migration, endocytosis, and development. The utility of the organism is dependent on the feasibility of genetic manipulation. Here, we present methods to transfect Dictyostelium discoideum cells that overcome existing limitations of culturing cells in liquid media.

キイロタマホコリカビの選択に基づいて細胞と細菌の成長の遺伝子工学

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other ...

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely related biochemical pathways. Previous methods such as the Synthetic Genetic Array (SGA) analysis, and random mutagenesis techniques using ultraviolet (UV) or chemicals like ethyl methanesulfonate (EMS) or N-ethyl-N- nitrosourea (ENU), have been widely used but are often costly and laborious. Also, these mutagen-based screening methods are frequently associated with severe side effects on the organism, inducing multiple mutations that add to the complexity of isolating the suppressors. Here, we present a simple and effective protocol to identify suppressor mutations in mutants which confer a growth defect in Schizosaccharomyces pombe. The fitness of cells with a growth deficiency in standard rich liquid media or synthetic liquid media can be monitored for recovery using an automated 96-well plate reader over an extended period. Once a cell acquires a suppressor mutation in the culture, its descendants outcompete those of the parental cells. The recovered cells that have a competitive growth advantage over the parental cells can then be isolated and backcrossed with the parental cells. The suppressor mutations are then identified using whole-genome sequencing. Using this approach, we have successfully isolated multiple suppressors that alleviate the severe growth defects caused by loss of Elf1, an AAA+ family ATPase that is important in nuclear mRNA transport and maintenance of genomic stability. There are currently over 400 genes in S. pombe with mutants conferring a growth defect. As many of these genes are uncharacterized, we propose that our method will hasten the identification of novel functional interactions with this user-friendly, high-throughput approach.

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

We present a simple suppressor screen protocol in fission yeast. This method is efficient, mutagen-free, and&#160; selective&#160; for&#160; mutations that&#160; often&#160; occur at&#160; &#160;a&#160; single&#160; genomic&#160; locus.&#160; The protocol is suitable for isolating suppressors that alleviate growth defects in liquid culture that are caused by a mutation or a drug.

分裂酵母分裂酵母のディープ シーケンス アシスト、自発的な抑制画面

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely ...

Semiconductor Sequencing for Preimplantation Genetic Testing for Aneuploidy

Chromosomal aneuploidy, one of the main causes leading to embryonic development arrest, implantation failure, or pregnancy loss, has been well documented in human embryos. Preimplantation genetic testing for aneuploidy (PGT-A) is a genetic test that significantly improves reproductive outcomes by detecting chromosomal abnormalities of embryos. Next-generation sequencing (NGS) provides a high-throughput and cost-effective approach for genetic analysis and has shown clinical applicability in PGT-A. Here, we present a rapid and low-cost semiconductor sequencing-based NGS method for screening of aneuploidy in embryos. The first step of the workflow is whole genome amplification (WGA) of the biopsied embryo specimen, followed by construction of sequencing library, and subsequent sequencing on the semiconductor sequencing system. Generally, for a PGT-A application, 24 samples can be loaded and sequenced on each chip generating 60&#8722;80 million reads at an average read length of 150 base pairs. The method provides a refined protocol for performing template amplification and enrichment of sequencing library, making the PGT-A detection reproducible, high-throughput, cost-efficient, and timesaving. The running time of this semiconductor sequencer is only 2&#8722;4 hours, shortening the turnaround time from receiving samples to issuing reports into 5 days. All these advantages make this assay an ideal method to detect chromosomal aneuploidies from embryos and thus, facilitate its wide application in PGT-A.

Here, we introduce a semiconductor sequencing method for preimplantation genetic testing for aneuploidy (PGT-A) with the advantages of short turnaround time, low cost, and high throughput.

無呼吸性遺伝子検査用半導体シーケンシング

Chromosomal aneuploidy, one of the main causes leading to embryonic development arrest, implantation failure, or pregnancy loss, has been well documented ...

Pooled CRISPR-Based Genetic Screens in Mammalian Cells

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian cells, this technology represents a novel means to perform genome-wide genetic screens for functional genomics studies. Libraries of guide RNAs (sgRNA) targeting all open reading frames permit the facile generation of thousands of genetic perturbations in a single pool of cells that can be screened for specific phenotypes to implicate gene function and cellular processes in an unbiased and systematic way. CRISPR-Cas screens provide researchers with a simple, efficient, and inexpensive method to uncover the genetic blueprints for cellular phenotypes. Furthermore, differential analysis of screens performed in various cell lines and from different cancer types can identify genes that are contextually essential in tumor cells, revealing potential targets for specific anticancer therapies. Performing genome-wide screens in human cells can be daunting, as this involves the handling of tens of millions of cells and requires analysis of large sets of data. The details of these screens, such as cell line characterization, CRISPR library considerations, and understanding the limitations and capabilities of CRISPR technology during analysis, are often overlooked. Provided here is a detailed protocol for the successful performance of pooled genome-wide CRISPR-Cas9 based screens.

CRISPR-Cas9 technology provides an efficient method to precisely edit the mammalian genome in any cell type and represents a novel means to perform genome-wide genetic screens. A detailed protocol discussing the steps required for the successful performance of pooled genome-wide CRISPR-Cas9 screens is provided here.

哺乳類細胞におけるCRISPRベースの遺伝子スクリーンのプール

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian ...

Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae). Together with 4 additional species, it forms the only known non-legume lineage able to establish a nitrogen-fixing nodule symbiosis with rhizobium. Comparative studies between legumes and P. andersonii could provide valuable insight into the genetic networks underlying root nodule formation. To facilitate comparative studies, we recently sequenced the P. andersonii genome and established Agrobacterium tumefaciens-mediated stable transformation and CRISPR/Cas9-based genome editing. Here, we provide a detailed description of the transformation and genome editing procedures developed for P. andersonii. In addition, we describe procedures for the seed germination and characterization of symbiotic phenotypes. Using this protocol, stable transgenic mutant lines can be generated in a period of 2-3 months. Vegetative in vitro propagation of T0 transgenic lines allows phenotyping experiments to be initiated at 4 months after A. tumefaciens co-cultivation. Therefore, this protocol takes only marginally longer than the transient Agrobacterium rhizogenes-based root transformation method available for P. andersonii, though offers several clear advantages. Together, the procedures described here permit P. andersonii to be used as a research model for studies aimed at understanding symbiotic associations as well as potentially other aspects of the biology of this tropical tree.

Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae) and can form nitrogen-fixing root nodules in association with the rhizobium. Here, we describe a detailed protocol for reverse genetic analyses in P. andersonii based on Agrobacterium tumefaciens-mediated stable transformation and CRISPR/Cas9-based genome editing.

窒素固定熱帯カンナバ科の木パラスポニア・アンダーソンニの変形、ゲノム編集、表現型

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae). Together with 4 additional species, it forms the ...