Name: biolistic transformation of a fluorescent tagged gene into the opportunistic fungal pathogen cryptococcus neoformans
Uploaded: 2015-03-19T16:00:00.000+00:00
Duration: 7 min 32 s
Description: Watch this Scientific Journal Video about Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans at JoVE.com

この作品は、国立科学財団（賞＃0920274）とサウスカロライナ州の試験場プロジェクトSC-1700340からの賞によってサポートされていました。クレムソン大学試験場の本論文isTechnical貢献号6283。著者は、原稿の彼らの重要な読書のためのこの最後のプロトコルおよび博士シェリルイングラム·スミス、ケイティグレン、とグレースKisirkoiの開発に彼の有益な助言のために博士はLukasz Kozubowskiに感謝。

注：このプロトコルの全体的なスキームを図1に概説されている。 1. C.ネオフォルマンスの準備<ol><li>各形質転換反応のために、C. 2〜3mlのO / N培養を成長さ250 rpmで振盪30℃でYPD培地でネオフォルマンス 。 </li><li> 10℃で900×gで5分間のO / N培養を遠心し、上清を捨てる。 </li><li>酵母ペプトンデキストロース（YPD）培地300μlの各細胞ペレットを再懸濁します。 </li><li>ガラスビーズを用いて、穏やかに1 Mソルビトールを含むYPD寒天上に洗浄し、細胞懸濁液を300μlを広げる。 </li><li>プレートは3〜4時間、周囲温度で乾燥することができます。 </li></ol> 2.ゴールドマイクロキャリアの準備<ol><li>ビーズをペレット、上清を除去してのddH 2 O、900×gで1分間遠心機の1ミリリットル中に0.6μmの金のビーズの0.25グラムを懸濁します。 </li><li> 100％エタノール1ml中の金ビーズを再懸濁する。&lt;/ LI&gt; <li> 4チューブ、250μlのそれぞれにビーズを配布し、100％エタノール750μlを添加する。 </li><li> 4℃で保存ゴールドビーズのアリコート。 </li></ol> 3. DNAの準備<ol><li>鉗子を用いて100％エタノールでそれらを浸漬することによってオレンジマクロキャリアバイオリスティックディスクを準備します。乾燥するDRIERITEを含む大きなペトリ皿に置きディスク（DRIERITEがディスクに接触していないことを確認してください）​​。 </li><li>乾燥したら、（以前は100％エタノールで拭い）銀ディスクホルダーにマクロキャリアディスクを押してください。 </li><li>渦ゴールドビーズ（ステップ2のように調製）とアリコート1.5ミリリットルマイクロ遠心チューブに12μlの、変態ごとに1つの管。 </li><li>順番に各チューブに入れる：2μgのDNA（好ましくは1μgのDNA /μlと2μL）、10μlの2.5 MのCaCl 2、および2μlの1 Mスペルミジン遊離塩基。 </li><li>ステップ3.4のように、しかし、誰DNAと負の制御を設定します。 </li><li>周囲温度でボルテックス各チューブとインキュベート5分。静かにこのインキュベーション中に定住ビーズを再懸濁し、時々各チューブをフリック。 </li><li> DNA被覆金ビーズをペレットに30秒間225×gでチューブをスピン。慎重に（ペッティングまたは吸引により）、上清を除去し、廃棄する。 </li><li>ゆっくり上下にピペッティングすることによって完全に100％エタノール600μlの再懸濁ビーズ。 </li><li>梱包せずにビーズをペレットに30秒間225×gでチューブをスピン。慎重に取り外し、上清を捨てる。 </li><li>ゆっくり上下にピペッティングすることによって100％エタノールの8μlのDNA被覆金ビーズを再懸濁します。 </li><li>直径1cmにおけるバイオリスティック、ディスクの中心上にDNA被覆金ビーズをピペット乾燥させます。 注：バイオリスティックディスクの中央に見える乾燥した金の円はゴールドビーズの十分な濃度が存在することを示す。 注：DNA被覆金ビーズでロードマクロキャリアディスクは現在、遺伝子銃で使用するための準備ができている。 </li></ol> 4. O遺伝子銃をperating <ol><li>真空ポンプの電源をオンにします。 </li><li>約2200 PSIの圧力は圧力計に到達するまで反時計回りにノブを回して、ヘリウムガスをオンにします。 </li><li>左側の赤いスイッチを切り替えることにより、遺伝子銃をオンにします。 </li><li>真空が15秒以内に28インチHgのに到達しますので、真空ベントのための流量が調整されていることを確認してください。 </li><li>破裂板とマクロキャリアとの間の距離が約3/8インチであることを確認してください。 </li><li>エタノールでダウン拭いてチャンバ全体を清掃してください。 </li><li> 100％エタノール中に破裂ディスクを沈める。無菌の表面（ 例えば 、ペトリ皿）上で乾燥することができます。 </li><li>ラプチャーディスクホルダーを緩めトルクレンチを使用してください。ホルダーにきれいな破裂ディスクを挿入します。元の位置に戻しラプチャーディスクホルダーをねじ込み、右に一度に回してトルクレンチで締めます。 注：破断ディスクは、各シュート以下の置換されます。 </li><li>目を沈める100％エタノールで電子メッシュスクリーン。無菌の表面（ 例えば 、ペトリ皿）上で乾燥することができます。 </li><li>乾燥したら、白いプラスチック取り付けプレート上で洗浄メッシュのスクリーンを配置。ダウンディスク室へのマクロキャリアディスクホルダのDNA側に配置します。銀キャップのネジ、そして最高のスロットに取り付けプレートを配置。 注：メッシュスクリーンは、それぞれの撮影の後に置き換えられます。 </li><li>底板上の1Mソルビトールを含むYPD寒天プレートを置きます。 </li><li>室のドアをシャットダウンして、所定の位置に固定。 </li><li>真空に係合し、真空28インチHgに達することができるように真ん中の赤いスイッチを押して、保持します。適切な真空レベルに達すると、上下の位置にこのスイッチを移動させる。準備ができたら、火に右側の赤いスイッチを押したままにします。ラプチャーディスクが表示されたら、すぐに発射ボタンを離すと0 PSIにチャンバーを排気する中間位置にミドル赤いスイッチを押してください。 </li><li>破裂板の破片を一掃し、遺伝子銃をオフにしてください。その後heliuをオフにするノブを時計回りに回し、最後に、真空ポンプをオフにすることで、Mガス。 </li></ol> 5.めっき形質転換細胞<ol><li>形質転換プレートは、細胞が回復できるように4時間室温で静置する。 </li><li>プレート上YPDのピペットで700μlの。優しく無菌1.5mlマイクロチューブに寒天とピペット液体のオフ細胞をこすり取るために、セルスクレーパーを使用してください。すべての細胞がプレートから回収されたことを確認するために、この手順を繰り返します。 </li><li> 30秒間225×gで細胞をペレット。上清を除去し、廃棄する。 </li><li> YPD500μlにペレットを再懸濁。 </li><li> YPD +抗生物質プレートの中心への細胞懸濁液をピペットで100μlのガラスビーズを用いて広げた。 </li><li> 3-4日室温で反転プレートのままにしておきます。コロニーが出現すると、新しいYPD +抗生物質プレート上にパッチ。 </li></ol> PCR用6.ゲノムDNAの単離注：これは、試薬を使用して修正バージョンですDNA精製キット（材料の表を参照してください）​​からのS。 <ol><li> Cの各5mlの文化を育てる30℃でYPD液体におけるネオフォルマンス形質転換体を 250 rpmでO / Nで振盪。 </li><li> 900×gで細胞の3ミリリットルをペレット、および核溶解液の600μlの中で再懸濁します。 </li><li> 0.5ミリメートル酸200μlで新しい1.5 mlのマイクロ遠心チューブにサスペンションを追加したガラスビーズを洗浄した。 </li><li>氷の上で周囲温度、クールチューブでのミニビードビーターで45秒間ホモジナイズし、繰り返します。 </li><li>サンプルは2分間氷上に落ち着くと新しい1.5mlチューブに上清を転送することができます。 20秒間激しくボルテックス（上清回復のすべての600μlの100μl）を、各チューブにタンパク質沈殿溶液200μlを追加します。 </li><li>サンプルは5分間氷上で落ち着く、と3分間11000×gで遠心分離することを許可する。 </li><li> RTイソプロパノール300μlのを含む新しい1.5 mlチューブに上清を移し。緩やかに転倒混和します。 </li><li>2分間11000×gで遠心サンプルは、注意深く上清を除去し、ペーパータオルの上にチューブを排出。 </li><li>各チューブに、RTの70％エタノール300μlを加え、ゆっくりペレットを洗浄する反転。 </li><li>遠心2分間11000×gでサンプル、慎重にエタノールをすべて削除する。 </li><li>清潔なペーパータオルの上にチューブを逆さにし、10〜15分間空気乾燥ペレットを許可する。 </li><li> DNAの再水和溶液50μlと各ペレットと渦へのRNase溶液1.5mlを添加する。 </li><li> 5秒間遠心分離したサンプルは、キャップから液体のすべてを削除します。 </li><li> 15分間37℃でサンプルをインキュベートする。 </li><li> 1時間65℃でサンプルをインキュベートすることによってDNAを再水和する。 </li><li> 260nmでの吸光度を測定することによって分光光度的にDNAを定量（1.0のA 260読み取りが〜50μg/ mlの二本鎖DNAと同等である）、各PCR反応には200ngまで使用。 </li></ol> 7. RNA単離のために転写酵素PCRリバース。 <ol><li> RNA精製キット（材料の表を参照してください）​​を使用し、minibeadbeaterを使用して酵母細胞からRNAを単離するために、メーカーの指示に従ってください。 </li><li> （1.0のA 260読み取りが約40 / mlの一本鎖RNAと等価である）260nmの吸光度を測定することにより、RNAの濃度を定量する。 </li><li> RT-PCRキット（材料の表を参照してください）​​を使用し、約1μgのRNAのでRT-PCR反応をセットアップするために、製造元の指示に従ってください。この研究で得られた結果は、 表1に列挙されたプライマーを使用する。 </li></ol>

<ol>
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We have nothing to disclose.

Utilizing this protocol, biolistic transformation can be accomplished in which linear DNA is integrated into a desired locus in the Cryptococcus neoformans genome by homologous recombination. Certain steps in the protocol can have a dramatic effect on the effectiveness/efficiency of the transformation. For a successful transformation, it is imperative that the DNA utilized in the shoot has a concentration of at least 1 &#181;g. However, the volume of DNA added to the gold beads can be increased in the chance the...

Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis resulting in more than 625,000 deaths per year worldwide 1. Acetate has been shown to be a major fermentation product during cryptococcal infection 2,3,4, and genes encoding enzymes from three putative acetate-producing pathways have been shown to be upregulated during infection 5. This suggests that acetate production and transport may be a necessary and required part of the pathogenic process; however, the significance of this is not yet understood. One possible pathway for acetate production is the xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp) - acetate kinase (Ack), a pathway previously thought to be present only in bacteria but recently identified in both euascomycete as well as basidiomycete fungi, including&#160;C. neoformans&#160;6.
To determine the localization of these enzymes of this pathway in the cell, a construct carrying a neomycin resistance gene downstream of an ACK gene fusion to the fluorescent tag mCherry (ACK:mCherry:Neo) will be introduced into C. neoformans using the well-established method of biolistic transformation 7,8. Although electroporation is an efficient method for transformation of plasmids that will be maintained as episomes into Cryptococcus 9, it is not useful in creating stable homologous transformants 8. Only&#160;biolistic&#160;delivery using a gene gun&#160;provides&#160;an effective means to transform&#160;linear&#160;DNAs&#160;that will be integrated into the genome by homologous recombination. For example, Edman et al. showed that of the transformants resulting from electroporation of a plasmid-borne URA5 selectable marker into C. neoformansura5 mutants, just 0.001 to 0.1% of transformants were stable 9. Chang et al. achieved just a 0.25% stable transformation efficiency using electroporation to reconstitute capsule production in an acapsular mutant 10. Unlike electroporation, biolistic transformation has been shown to result in stable transformation efficiency of 2-50% depending on the gene that is being altered 7,8,11. 
This visual experiment will provide a step-by-step demonstration of biolistic transformation of the linear ACK:mCherry:Neo DNA construct into C. neoformans, and will describe how to confirm its proper integration via homologous recombination into the&#160;ack&#160;locus. The protocol demonstrated here is a modification of the method developed in the Perfect laboratory 8.

<table><tbody><tr><td>0.6 &amp;mu;m gold beads</td><td>Bio-Rad</td><td>165-2262</td><td>http://www.bio-rad.com</td></tr><tr><td>Spermadine-free base</td><td>Sigma- Aldrich</td><td>S0266</td><td>https://www.sigmaaldrich.com</td></tr><tr><td>G418 - Sulfate (Neomycin)</td><td>Gold Biotechnology</td><td>G-418-10</td><td>www.goldbio.com</td></tr><tr><td>Hygromycin</td><td>Gold Biotechnology</td><td>H-270-1</td><td>www.goldbio.com</td></tr><tr><td>1350 psi Rupture Discs</td><td>Bio-Rad</td><td>165-2330</td><td>http://www.bio-rad.com</td></tr><tr><td>Stopping Screens</td><td>Bio-Rad</td><td>165-2336</td><td>http://www.bio-rad.com</td></tr><tr><td>Macrocarriers discs</td><td>Bio-Rad</td><td>165-2335</td><td>http://www.bio-rad.com</td></tr><tr><td>YPD Broth</td><td>Becton Dickinson &amp;amp; Co.</td><td>242820</td><td>www.bd.com</td></tr><tr><td>Agar</td><td>Becton Dickinson &amp;amp; Co.</td><td>214530</td><td>www.bd.com</td></tr><tr><td>Sorbitol</td><td>Fisher Scientific</td><td>BP439</td><td>http://www.fishersci.com</td></tr><tr><td>PDS-1000/He System</td><td>Bio-Rad</td><td>165-2257</td><td>http://www.bio-rad.com</td></tr><tr><td>Microscope</td><td>Zeiss</td><td>Axio</td><td>http://www.zeiss.com/microscopy</td></tr><tr><td>KOD One Step PCR Kit</td><td>EMD Millipore</td><td>71086-4</td><td>http://www.emdmillipore.com</td></tr><tr><td>One Step RT-PCR Kit</td><td>Qiagen</td><td>210212</td><td>www.qiagen.com</td></tr><tr><td>Wizard Genomic DNA Purification Kit</td><td>Promega</td><td>A1120</td><td>www.promega.com</td></tr><tr><td>RNeasy Mini Kit</td><td>Qiagen</td><td>74104</td><td>www.qiagen.com</td></tr><tr><td>Mini Beadbeater - 1</td><td>BioSpecs</td><td>3110BX</td><td>http://www.biospec.com</td></tr><tr><td>Microfuge 18 Centrifuge</td><td>Beckman Coulter</td><td>F241.5P</td><td>www.beckmancoulter.com</td></tr><tr><td>Microplate Spectrophotometer</td><td>BioTek</td><td>EPOCH</td><td>www.biotek.com</td></tr></tbody></table>

biolistic transformation of a fluorescent tagged gene into the opportunistic fungal pathogen cryptococcus neoformans

担子菌クリプトコッカスネオフォルマンス 、中枢神経系の侵襲的な日和見病原体は、世界的に全世界で年間以上の625000人が死亡、その結果、真菌性髄膜炎の最も頻繁な原因である。エレクトロポレーションは、 クリプトコッカスにおけるプラスミドの形質転換のために開発されてきたが、唯一のバイオリスティック送達は、相同組換えによりゲノム中に組み込むことができる直鎖状DNAを形質転換する有効な手段を提供する。 酢酸クリプトコッカス感染中の主要な発酵産物であることが示されているが、この重要性はまだ知られていない。酵素がキシルロース-5-リン酸/フルクトース-6-リン酸ホスホケトラーゼ（XFP）及び酢酸キナーゼ（肯定応答）からなる細菌の経路はC.で酢酸を製造するための3つの潜在的な経路の一つであるネオフォルマンス 。ここでは、構築物の微粒子銃形質転換を実証する、のAckをコードする遺伝子は、mCherryを蛍光タグに融合C.にしたネオフォルマンス 。次に、ACK座にACK -mCherry融合の統合を確認する。

C.の成功バイオリスティック変換ネオフォルマンス、このプロトコル方式（ 図1）に従って求めることができる。 DNAは（ 図2A）撮影された後にバイオリスティック形質転換と、コーティングされた金のビーズの成功したシュートは、プレート上に見えるゴールドリングで示されている。選択培地上にYPD + 1Mソルビトールプレートから回収した細胞をプレ?...

Watch this Scientific Journal Video about Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans at JoVE.com

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

バイオリスティック形質転換は、相同組換えによる日和見病原体クリプトコッカス·ネオフォルマンスのゲノムへのDNAの安定な組み込みを生成するために使用される方法である。我々 は 、C。 ネオフォルマンスへの蛍光タグmCherryをに融合をコードする遺伝子の酢酸キナーゼを持つ構築物のバイオリスティック形質転換のデモンストレーションを行います。

日和見菌病原体への蛍光タグ付き遺伝子のバイオリスティック変容<em&gt;クリプトコックス·ネオフォルマンス</em

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

biolistic-transformation-fluorescent-tagged-gene-into-opportunistic

Research

JoVE Journal

Immunology and Infection

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

9.9K ビュー.  Clemson University. バイオリスティック形質転換は、相同組換えによる日和見病原体クリプトコッカス·ネオフォルマンスのゲノムへのDNAの安定な組み込みを生成するために使用される方法である。我々 は 、C。 ネオフォルマンスへの蛍光タグmCherryをに融合をコードする遺伝子の酢酸キナーゼを持つ構築物のバイオリスティック形質転換のデモンストレ?...

ビデオ: Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Generation of Fluorescent Protein Fusions in Candida Species

Candida species, prevalent colonizers of the intestinal and genitourinary tracts, are the cause of the majority of invasive fungal infections in humans. Thus, molecular and genetic tools are needed to facilitate the study of their pathogenesis mechanisms. PCR-mediated gene modification is a straightforward and quick approach to generate epitope-tagged proteins to facilitate their detection. In particular, fluorescent protein (FP) fusions are powerful tools that allow visualization and quantitation of both yeast cells and proteins by fluorescence microscopy and immunoblotting, respectively. Plasmids containing FP encoding sequences, along with nutritional marker genes that facilitate the transformation of Candida species, have been generated for the purpose of FP construction and expression in Candida. Herein, we present a strategy for constructing a FP fusion in a Candida species. Plasmids containing the nourseothricin resistance transformation marker gene (NAT1) along with sequences for either green, yellow, or cherry FPs (GFP, YFP, mCherry) are used along with primers that include gene-specific sequences in a polymerase chain reaction (PCR) to generate a FP cassette. This gene-specific cassette has the ability to integrate into the 3'-end of the corresponding gene locus via homologous recombination. Successful in-frame fusion of the FP sequence into the gene locus of interest is verified genetically, followed by analysis of fusion protein expression by microscopy and/or immuno-detection methods. In addition, for the case of highly expressed proteins, successful fusions can be screened for primarily by fluorescence imaging techniques.

Generation of Fluorescent Protein Fusions in Candida Species

PCR-mediated gene modification can be used to generate fluorescent protein fusions in Candida species, which facilitates visualization and quantitation of yeast cells and proteins. Herein, we present a strategy for constructing a fluorescent protein fusion (Eno1-FP) in Candida parapsilosis.

蛍光タンパク質融合物中の生成<em&gt;カンジダ</em&gt;種

Candida species, prevalent colonizers of the intestinal and genitourinary tracts, are the cause of the majority of invasive fungal infections in humans. ...

遺伝学

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some factors act directly on virulence genes; others control pathogenesis by adjusting the expression of downstream regulators or the accumulation of signals that affect regulator activity. While regulation has been studied extensively during in vitro growth, relatively little is known about how gene expression is adjusted during infection. Such information is important when a particular gene product is a candidate for therapeutic intervention. Transcriptional approaches like quantitative, real-time RT-PCR and RNA-Seq are powerful ways to examine gene expression on a global level but suffer from many technical challenges including low abundance of bacterial RNA compared to host RNA, and sample degradation by RNases. Evaluating regulation using fluorescent reporters is relatively easy and can be multiplexed with fluorescent proteins with unique spectral properties. The method allows for single-cell, spatiotemporal analysis of gene expression in tissues that exhibit complex three-dimensional architecture and physiochemical gradients that affect bacterial regulatory networks. Such information is lost when data are averaged over the bulk population. Herein, we describe a method for quantifying gene expression in bacterial pathogens in situ. The method is based on simple tissue processing and direct observation of fluorescence from reporter proteins. We demonstrate the utility of this system by examining the expression of Staphylococcus aureus thermonuclease (nuc), whose gene product is required for immune evasion and full virulence ex vivo and in vivo. We show that nuc-gfp is strongly expressed in renal abscesses and reveal heterogeneous gene expression due in part to apparent spatial regulation of nuc promoter activity in abscesses fully engaged with the immune response. The method can be applied to any bacterium with a manipulatable genetic system and any infection model, providing valuable information for preclinical studies and drug development.

Described here is a method for analyzing bacterial gene expression in animal tissues at a cellular level. This method provides a resource for studying the phenotypic diversity occurring within a bacterial population in response to the tissue environment during an infection.

感染した組織の細菌の遺伝子の規則を勉強する蛍光ベースのメソッド

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some ...

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended intracellular endotubes that build and stabilize the lumen with a membrane and submembraneous cytoskeleton of apical character. The excretory cell expands its length approximately 2,000 times to generate these canals, making this model unique for the in vivo assessment of de novo polarized membrane biogenesis, intracellular lumen morphogenesis and unicellular tubulogenesis. The protocol presented here shows how to combine standard labeling, gain- and loss-of-function genetic or RNA interference (RNAi)-, and microscopic approaches to use this model to visually dissect and functionally analyze these processes on a molecular level. As an example of a labeling approach, the protocol outlines the generation of transgenic animals with fluorescent fusion proteins for live analysis of tubulogenesis. As an example of a genetic approach, it highlights key points of a visual RNAi-based interaction screen designed to modify a gain-of-function cystic canal phenotype. The specific methods described are how to: label and visualize the canals by expressing fluorescent proteins; construct a targeted RNAi library and strategize RNAi screening for the molecular analysis of canal morphogenesis; visually assess modifications of canal phenotypes; score them by dissecting fluorescence microscopy; characterize subcellular canal components at higher resolution by confocal microscopy; and quantify visual parameters. The approach is useful for the investigator who is interested in taking advantage of the C. elegans excretory canal for identifying and characterizing genes involved in the phylogenetically conserved processes of intracellular lumen and unicellular tube morphogenesis.

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The C. elegans excretory canal is a unique single-cell model for the visual in vivo analysis of de novo polarized membrane biogenesis. This protocol describes a combination of standard genetic/RNAi and imaging approaches, adaptable for the identification and characterization of molecules directing unicellular tubulogenesis, and apical membrane and lumen biogenesis.

細胞内の内腔の形態形成と単一細胞におけるIn Vivo偏光膜生合成のためのモデルとして線虫排泄管: GFP 融合、RNAi 操作画面やイメージングによるラベリング

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended ...

発生生物学

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic redundancy affecting simple genetics in higher plants. The novel model organism Ostreococcus tauri is the smallest free-living eukaryote known to date, and possesses a greatly reduced genome size and cellular complexity1,2, manifested by the presence of just one of most organelles (mitochondrion, chloroplast, golgi stack) per cell, and a genome containing only ~8000 genes. Furthermore, the combination of unicellularity and easy culture provides a platform amenable to chemical biology approaches. Recently, Ostreococcus has been successfully employed to study basic mechanisms underlying circadian timekeeping3-6. Results from this model organism have impacted not only plant science, but also mammalian biology7. This example highlights how rapid experimentation in a simple eukaryote from the green lineage can accelerate research in more complex organisms by generating testable hypotheses using methods technically feasible only in this background of reduced complexity. Knowledge of a genome and the possibility to modify genes are essential tools in any model species. Genomic1, Transcriptomic8, and Proteomic9 information for this species is freely available, whereas the previously reported methods6,10 to genetically transform Ostreococcus are known to few laboratories worldwide.
In this article, the experimental methods to genetically transform this novel model organism with an overexpression construct by means of electroporation are outlined in detail, as well as the method of inclusion of transformed cells in low percentage agarose to allow selection of transformed lines originating from a single transformed cell. Following the successful application of Ostreococcus to circadian research, growing interest in Ostreococcus can be expected from diverse research areas within and outside plant sciences, including biotechnological areas. Researchers from a broad range of biological and medical sciences that work on conserved biochemical pathways may consider pursuing research in Ostreococcus, free from the genomic and organismal complexity of larger model species.

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

This article describes genetic transformation of the unicellular marine alga Ostreococcus tauri by electroporation. This eukaryotic organism is an effective model platform for higher plants, possesing greatly reduced genomic and cellular complexity and being readily amenable to both cell culture and chemical biology.

Picoeukaryoteのゲノム形質転換<em&gt; Ostreococcusタウリ</em

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic ...

生物学

Agrobacterium tumefaciensを用いたChlorella vulgarisの安定的な遺伝子統合

Agrobacterium tumefaciens-Mediated Genetic Engineering of Green Microalgae, Chlorella vulgaris

Agrobacterium tumefaciens-mediated transformation (AMT) serves as a widely employed tool for manipulating plant genomes. However, A. tumefaciens exhibit the capacity for gene transfer to a diverse array of species. Numerous microalgae species lack well-established methods for reliably integrating genes of interest into their nuclear genome. To harness the potential benefits of microalgal biotechnology, simple and efficient genome manipulation tools are crucial. Herein, an optimized AMT protocol is presented for the industrial microalgae species Chlorella vulgaris, utilizing the reporter green fluorescent protein (mGFP5) and the antibiotic resistance marker for Hygromycin B. Mutants are selected through plating on Tris-Acetate-Phosphate (TAP) media containing Hygromycin B and cefotaxime. Expression of mGFP5 is quantified via fluorescence after over ten generations of subculturing, indicating the stable transformation of the T-DNA cassette. This protocol allows for the reliable generation of multiple transgenic C. vulgaris colonies in under two weeks, employing the commercially available pCAMBIA1302 plant expression vector.

著者スポットライト:Agrobacterium tumefaciensを使用したChlorella vulgarisの最適化された形質転換プロトコル 

This protocol outlines the utilization of Agrobacterium tumefaciens-mediated transformation (AMT) for integrating gene(s) of interest into the nuclear genome of the green microalgae Chlorella vulgaris, leading to the production of stable transformants.

Agrobacterium tumefaciens-緑色微細藻類Chlorella vulgarisの遺伝子工学

Agrobacterium tumefaciens-mediated transformation (AMT) serves as a widely employed tool for manipulating plant genomes. However, A. tumefaciens exhibit ...

生物工学

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a fluorescent reporter. The constantly evolving list of genetically encoded fluorescent proteins (FPs) presents users with several alternatives when it comes to fluorescent fusion design. Each FP has specific optical and biophysical properties that can affect the biochemical, cellular, and functional properties of the resulting fluorescent fusions. For instance, several FPs tend to form nonspecific oligomers that are susceptible to impede on the function of the fusion partner. Unfortunately, only a few methods exist to test the impact of FPs on the behavior of the fluorescent reporter. Here, we describe a simple method that enables the rapid assessment of the impact of FPs using polyglutamine (polyQ) toxicity assays in the budding yeast Saccharomyces cerevisiae. PolyQ-expanded huntingtin proteins are associated with the onset of Huntington&#39;s disease (HD), where the expanded huntingtin aggregates into toxic oligomers and inclusion bodies. The aggregation and toxicity of polyQ expansions in yeast are highly dependent on the sequences flanking the polyQ region, including the presence of fluorescent tags, thus providing an ideal experimental platform to study the impact of FPs on the behavior of their fusion partner.

This article describes protocols to assess the effect of fluorescent proteins on the aggregation and toxicity of misfolded polyglutamine expansion for the rapid evaluation of a newly uncharacterized fluorescent protein in the context of fluorescent reporters.

酵母におけるポリグルタミン毒性の試金を使用して融合パートナーの蛍光タンパク質の効果

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...

Small-Scale Plasma Membrane Preparation for the Analysis of Candida albicans Cdr1-mGFPHis

The successful biochemical and biophysical characterization of ABC transporters depends heavily on the choice of the heterologous expression system. Over the past two decades, we have developed a yeast membrane protein expression platform that has been used to study many important fungal membrane proteins. The expression host Saccharomyces cerevisiae AD&#916;&#916; is deleted in seven major endogenous ABC transporters and it contains the transcription factor Pdr1-3 with a gain-of-function mutation that enables the constitutive overexpression of heterologous membrane protein genes stably integrated as single copies at the genomic PDR5 locus. The creation of versatile plasmid vectors and the optimization of one-step cloning strategies enables the rapid and accurate cloning, mutagenesis, and expression of heterologous ABC transporters. Here, we describe the development and use of a novel protease-cleavable mGFPHis double tag (i.e., the monomeric yeast enhanced green fluorescent protein yEGFP3 fused to a six-histidine affinity purification tag) that was designed to avoid possible interference of the tag with the protein of interest and to increase the binding efficiency of the His tag to nickel-affinity resins. The fusion of mGFPHis to the membrane protein ORF (open reading frame) enables easy quantification of the protein by inspection of polyacrylamide gels and detection of degradation products retaining the mGFPHis tag. We demonstrate how this feature facilitates detergent screening for membrane protein solubilization. A protocol for the efficient, fast, and reliable isolation of the small-scale plasma membrane preparations of the C-terminally tagged Candida albicans multidrug efflux transporter Cdr1 overexpressed in S. cerevisiae AD&#916;&#916;, is presented. This small-scale plasma membrane isolation protocol generates high-quality plasma membranes within a single working day. The plasma membrane preparations can be used to determine the enzyme activities of Cdr1 and Cdr1 mutant variants.

Small-Scale Plasma Membrane Preparation for the Analysis of Candida albicans Cdr1-mGFPHis

This article presents a small-scale plasma membrane isolation protocol for the characterization of Candida albicans ABC (ATP-binding cassette) protein Cdr1, overexpressed in Saccharomyces cerevisiae. A protease-cleavable C-terminal mGFPHis double tag with a 16-residue linker between Cdr1 and the tag was designed to facilitate the purification and detergent-screening of Cdr1.

カンジダ・アルビカンスCdr1-mGFPHisの分析のための小スケールプラズマ膜調製

The successful biochemical and biophysical characterization of ABC transporters depends heavily on the choice of the heterologous expression system. Over ...

Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna

Cyanobacteria are the focus of basic research and biotechnological projects in which solar energy is utilized for biomass production. Phormidium lacuna is a newly isolated filamentous cyanobacterium. This paper describes how new filamentous cyanobacteria can be isolated from marine rockpools. It also describes how DNA can be extracted from filaments and how the genomes can be sequenced. Although transformation is established for many single-celled species, it is less frequently reported for filamentous cyanobacteria. A simplified method for the natural transformation of P. lacuna is described here. P. lacuna is the only member of the order Oscillatoriales for which natural transformation is established. This paper also shows how natural transformation is used to express superfolder green fluorescent protein (sfGFP). An endogenous cpcB promoter induced approximately 5 times stronger expression than cpc560, A2813, or psbA2 promoters from Synechocystis sp. PCC6803. Further, a method for the cryopreservation of P. lacuna and Synechocystis sp. CPP 6803 was established, and methods for assessing motility in a liquid medium and on agar and plastic surfaces are described.

Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna

Phormidium lacuna is a filamentous cyanobacterium that was isolated from marine rockpools. This article describes the isolation of filaments from natural sources, DNA extraction, genome sequencing, natural transformation, expression of sfGFP, cryoconservation, and motility methods.

糸状シアノバクテリウム・ホルミジウム・ラクナの自然形質転換、タンパク質発現、凍結保存

Cyanobacteria are the focus of basic research and biotechnological projects in which solar energy is utilized for biomass production. Phormidium lacuna is ...

日和見菌病原体への蛍光タグ付き遺伝子のバイオリスティック変容

要約

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蛍光タンパク質融合物中の生成

感染した組織の細菌の遺伝子の規則を勉強する蛍光ベースのメソッド

細胞内の内腔の形態形成と単一細胞におけるIn Vivo偏光膜生合成のためのモデルとして線虫排泄管: GFP 融合、RNAi 操作画面やイメージングによるラベリング

Picoeukaryoteのゲノム形質転換

Agrobacterium tumefaciens-緑色微細藻類Chlorella vulgarisの遺伝子工学

Agrobacterium tumefaciens-緑色微細藻類Chlorella vulgarisの遺伝子工学

Agrobacterium tumefaciens-緑色微細藻類Chlorella vulgarisの遺伝子工学

酵母におけるポリグルタミン毒性の試金を使用して融合パートナーの蛍光タンパク質の効果

カンジダ・アルビカンスCdr1-mGFPHisの分析のための小スケールプラズマ膜調製

糸状シアノバクテリウム・ホルミジウム・ラクナの自然形質転換、タンパク質発現、凍結保存