ステップ1： レンチウイルスの生産のための293細胞とプレートを解凍。 <ol><li> 15 mlのチューブにDMEM +培地9mlのを準備します。 </li><li>液体窒素タンクから凍結293株のバイアルを取り出して、（すべてではない）ほとんどの細胞が融解するまで37℃の水浴中にバイアルを入れて。 </li><li>ステップ1から15ミリリットルチューブに凍結バイアルと場所から細胞を取り除きます。 </li><li> 5分間180グラムで遠心分離し、上清を捨てる。 </li><li> DMEM +培地10 mlで細胞を再懸濁し、そしてゼラチンでコーティングされた100 mmディッシュに移す。彼らは80〜90％コンフルエントになるまで37℃、5％CO 2インキュベーターで細胞をインキュベートする。 </li><li> 293細胞の継代：PBSで洗浄細胞、PBSを吸引除去、および0.05％trypsin/0.53 mMのEDTAの10cmの皿あたり4 mlを追加し、37℃で1分間インキュベート℃に</li><li> 10ミリリットルDMEM +培地で再懸濁し、タップして皿から細胞を剥離し、15 mlのチューブに移す。 5分間180グラムで遠心分離し、上清を吸引除去する。 </li><li> DMEM +培地の適切な量を追加し、数回上下にピペッティングして、単一の細胞懸濁液に細胞を壊す。 </li><li>セルの数をカウントし、FPの培地でmlあたり8 × 10 5細胞に濃度を調整する。 </li><li>種子の100 mm培養ディッシュあたり8 × 10 6個の細胞（10 ml）で、細胞、および37℃で一晩インキュベート° C、5％CO 2。 </li></ol>ステップ2： レンチウイルスプラスミドと収穫のウイルスを293細胞にトランスフェクション<ol><li>各10cmのプレートの場合は、FUW -テト- cDNAの10μgの（Oct4の、レトロウイルスベクター、KLF4またはc - Mycの）PMD - Gの2.5ugとレンチウイルスのバックボーンとpPax2の7.5μgを使用してください。 </li><li>チューブに3つのプラスミドを小分けしながら、血清を含まないDMEMの500μLを含む第2のチューブにFuGENE 6トランスフェクション試薬の30μlのを提供し、室温で5分間指のタッピングとインキュベートすることにより穏やかに混合する。 </li><li> DNA混合物をトランスフェ6/DMEM-containingチューブにドロップでドロップを追加し、20分間、指のタッピングとインキュベートすることにより穏やかに混合する。 </li><li> 293のの皿にDNA /のFugene 6複雑な滴下を追加し、37℃で一晩インキュベート° C、5％CO 2。 </li><li>翌朝：トランスフェクション試薬を含む培地を吸引、新鮮なES細胞培地5mlを追加し、インキュベーターにセルを返します。 </li><li> 48時間とトランスフェクション後72時間で、0.45μmのポアサイズの酢酸セルロースフィルターを通してそれをフィルタリング、および15 mlのチューブに移し、10 mlの滅菌使い捨て注射器を使って、293年代から培地を収集する。 </li><li>超遠心分離によりウイルスの上清を集中する。目的のボリュームにウイルスペレットを再懸濁し、Oct-3/4-、Sox2の - 、KLF4 -およびc - Myc -レンチウイルスを含む培地の等量のamixtureを行います。 </li><li>ウイルスを作成しているときに、10cmの皿（皿あたり2 × 10 6細胞）でコンフルエント90％にOct4-neo/rtTAまたはOct4-GFP/rtTA MEFSを（通路&lt;3）の準備</li><li>培地を吸引除去し、PBSの10mlで洗う。 </li><li> 0.05パーセントtrypsin/0.53 mMのEDTAのディッシュあたり1 mlを追加し、37℃で10分間、PBSを捨てます。 </li><li>培地9mlのを追加し、単一のセルにセルを一時停止し、15 mlのチューブに移す。 </li><li>セルの数をカウントし、mlあたり8 × 10 4個の細胞に濃度を調整する。ゼラチンでコーティングした10cmのディッシュに細胞懸濁液10ml（8 × 10 5細胞）を移します。一晩37皿をインキュベート° C、5％CO 2。 </li><li>線維芽細胞のディッシュから培地を吸引し、そしてウイルスを含む培地10mlを加え。 4時間から37℃で一晩に、細胞をインキュベート° C、5％CO 2を 。 </li><li> 24時間後線維芽細胞のディッシュから培地を吸引除去し、新鮮なES細胞用培地10mlを加え。 </li><li> 4つの遺伝子の発現を開始するためにドキシサイクリンを含む培地で、通常のES細胞用培地を交換し、他の言葉で、再プログラミング開始。 </li><li> Oct4は、ネオレポーターを使用している場合は、6と9日の間いつでも薬剤のセクションを開始する。 </li><li>コロニーをピックアップするのに十分な大きさになるまで、毎日培地を変更します。コロニーは、最初のプログラミングの開始後約週見えるようになります。彼らは20日目の周りにピックアップされるのに十分な大きさになるはず。 </li></ol>ステップ3： iPS細胞コロニーをピックアップピッキング前日： <ol><li> γ線照射DR4 MEFの持つゼラチンコートした24ウェルプレートを必要な数の種子</li></ol> 1日目のクローンをピッキング。 <ol><li> 1〜2時間取る前プレート上のコロニーを養う。 </li><li> V底96ウェルディッシュのウェルにPBS 15μlを添加する。 </li><li> PBSで一度ピックアップし、皿にPBS 10mlのを追加するコロニーを含む皿を洗ってください。 </li><li>小さな5μlのピペットチップ（4μlのP20 pipetmanを設定）を使用してコロニーを選択してください。転送CO96ウェルディッシュにもへロニーズ。 </li><li>よくマルチチャンネル（12）pipetorを使用してそれぞれにトリプシンを20μlを添加する。ピペッティング3回。 37℃で4分間。 </li><li>トリプシン処理コロニーにES培地100μlを加え。ピペッティング10回。 12位pipetor上の他のすべての位置にチップを用いて24ウェルディッシュに96ウェルディッシュから一度に6個のクローンを転送します。 </li><li>細胞はES細胞用培地を毎日供給、80〜90％コンフルエントに達するまで、37 ° C、5％CO 2インキュベーター内で24ウェルプレート上で細胞を成長させる。細胞は、おそらく3-7日で拡大できるようになります。 </li></ol>ステップ4 <ol><li> iPS細胞の拡大<ol><li>培地を吸引除去し、1mlのPBSで細胞を洗浄する。 </li><li>完全にPBSを除去、37℃で0.25％トリプシン/ 1mMのEDTAとインキュベート0.1 mlを加える℃で10分間。 </li><li> ES培地0.4 mlを加え、単一の細胞懸濁液にピペッティングにより細胞を一時停止。 </li><li> 6ウェルプレートのウェルに細胞懸濁液を転送、1.5 mlのES細胞用培地を追加し、そして細胞は6ウェルプレートでコンフルエント80〜90％に達するまで37℃、5％CO 2インキュベーター中でインキュベートする。この時点で、次のように、細胞の凍結ストックを準備。 </li></ol></li><li>凍結ストックの調製<ol><li>培地を吸引し、PBS 2mlで細胞を洗浄する。 </li><li>完全にPBSを除去、37℃で0.25％トリプシン/ 1mMのEDTAとインキュベートの0.5 mlを加える℃で10分間。 </li><li> ES培地2 mlを加え、単一の細胞懸濁液にピペッティングにより細胞を一時停止。 </li><li> 15 mlのチューブに細胞懸濁液を転送する、セルの数をカウントし、細胞をスピンダウン。 </li><li> 、上清を捨てmlあたり2 × 10 6個の細胞で濃度にES培地で細胞を懸濁します。 </li><li> 2 ES細胞の凍結培地（ES培地で20％のDMSO）とバイアル当たり0.5 mlのアリコートを、それを準備する。 </li><li>バイアルを凍結し、穏やかに混合して細胞懸濁液0.5 mlを移します。 </li><li>液体窒素に転送する長期保存のための次の日、細胞凍結容器にバイアルを入れ、-80、それを維持℃で一晩。 </li></ol></li></ol>

<ol>
	<li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126, 663-676 (2006).</li><li>Wernig, 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=et+al+.+In+vitro+reprogramming+of+fibroblasts+into+a+pluripotent+ES-cell-like+state.">et al . In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state.</a> Nature. 448, 318-324 (2007).</li><li>Okita, K., Ichisaka, T., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+germline-competent+induced+pluripotent+stem+cells.">Generation of germline-competent induced pluripotent stem cells.</a> Nature. 448, 313-317 (2007).</li><li>Maherali, N., 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=Global+epigenetic+remodeling+in+directly+reprogrammed+fibroblasts.">Global epigenetic remodeling in directly reprogrammed fibroblasts.</a> Cell Stem Cell. 1, 55-70 (2007).</li><li>Brambrink, T., 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=Sequential+expression+of+pluripotency+markers+during+direct+reprogramming+of+somatic+cells.">Sequential expression of pluripotency markers during direct reprogramming of somatic cells.</a> Cell Stem Cell. 2, 151-159 (2008).</li></ol>

このビデオでは、我々はOct4-GFP/R26-M2rtTAとNanog-GFP/R26-M2rtTAマウス由来のMEFからのGFP陽性、多能性iPS細胞を生成するために誘導、レンチウイルスシステムを使用する方法を示します。それはプログラミングプロセス中に導入遺伝子発現の制御が可能なため、この手順では、iPS細胞を生成するための有用な方法です。 iPS細胞の発生におけるウイルスの使用が臨床の現場でこれらの細胞の使用を妨げるが、この手順は、私たちはまだ定義されている更新プロセスを下線主要な生物学的な疑問の幾つかに解答したりすることができます。

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generating ips cells from mefs through forced expression of sox-2, oct-4, c-myc, and klf4

多能性は、Oct4のウイルスの形質導入により差別化されたマウスで誘導することができる、レトロウイルスベクター、KLF4、およびc - Myc（高橋と山中、2006; Wernig、ら、2007;。沖田ら、2007;。。Maherali、ら、 2007年）。我々は、これら4つの転写因子は、ドキシサイクリン（DOX）から発現されるプログラミング戦略を考案したレンチウイルスベクターを（Brambrinkら、2008）誘​​導。これらの誘導の構文を使用して、我々はマウス胚性繊維芽細胞（MEF）から誘導多能性幹（iPS）細胞を得ることができます。このビデオでは、我々は4つの転写因子を発現し、iPS細胞を生成するためにこれらのウイルスとMEFのを感染させる方法を示す誘導レンチウイルスを生成するための手順を示しています。誘導レンチウイルスを使用することにより、培地にdoxycylineを追加することにより制御の4つの因子の発現。従来のレトロウイルス感染に対するこのシステムの利点は、プログラミングと遺伝子発現の要件の動態を詳細に分析できるようにオフの遺伝子をオンとする機能です。

Watch this Scientific Journal Video about Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4 at JoVE.com

Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

このビデオでは、誘導レンチウイルスを用いて誘導多能性幹細胞を生成するための手順を示して発現するOct4は、レトロウイルスベクター、c - MycおよびKLF4。

ソックス- 2の強制発現によってMEFSからiPS細胞を生成し、OCT - 4、c - Mycの、とKLF4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006;  Wernig, et al., ...

generating-ips-cells-from-mefs-through-forced-expression-sox-2-oct-4

Research

JoVE Journal

Biology

36.1K ビュー.  Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology. このビデオでは、誘導レンチウイルスを用いて誘導多能性幹細胞を生成するための手順を示して発現するOct4は、レトロウイルスベクター、c - MycおよびKLF4。

ビデオ: Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

From MEFs to Matrigel I: Passaging hESCs in the Presence of MEFs

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells.


This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells. Part 1 of 3.

MEFの存在下で継代ヒトES細胞：MEFをから私をマトリゲルする

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells.
...

生物学

From MEFs to Matrigel 2: Splitting hESCs from MEFs onto Matrigel

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells, how to passage hESCs from MEF plates to feeder cell-free Matrigel plates.


This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 2 of 3.

マトリゲル上のMEFから分割ヒトES細胞：MEFのからマトリゲル2〜

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells, how to passage hESCs from MEF ...

From MEFs to Matrigel 3: Passaging hESCs from Matrigel onto Matrigel

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated.

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 3 of 3.

マトリゲル上にマトリゲルからの継代ヒトES細胞：MEFのからマトリゲル3〜

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

エレクトロポレーションのためのマウス骨髄からの一次造血細胞培養の準備

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

クーマシー有機溶媒および酢酸のないG - 250でゲル中のタンパク質の染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

高効率で初代培養細胞をトランスフェクトするためにジーンパルサーMXcellのエレクトロポレーションシステムの使用

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

ナマルバ細胞におけるIL - 4依存性遺伝子発現のsiRNAのノックダウン：遺伝子発現研究を簡素化する自動化セルカウンターの使い方

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS cell cultures to feeder-free while still maintaining your use of Knockout SR.

The following protocol provides instruction for adapting human induced Pluripotent Stem (iPS) Cells to feeder-free culture using complete KnockOut Serum Replacement Feeder-Free medium (KSR-FF). Once adapted, instructions for continual maintenance are also provided.

完全ノックアウト血清代替フィーダーを含まない培地を用いてヒトiPS細胞のフィーダーフリー適応、文化と継

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem ...

Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS and hES cell cultures to feeder-free while still maintaining your use of Knockout SR.

This protocol describes the detailed procedure for cryopreserving human iPS cells in KnockOut SR cryopreservation medium and recovering these cells in complete KnockOut SR Feeder Free (KSR-FF) medium or feeder-based KnockOut SR medium.

完全ノックアウト血清代替のフィーダーを含まない培地を用いてヒトiPS細胞のCryopreservingおよびリカバリ

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

ヒト細胞から分泌タンパク質の生産のための便利で一般式プラットフォーム

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...