サンドラー喘息基礎研究センター（SABRE） サンドラー財団

パート1：レンチウイルスを生成するためにHEK 293細胞のトランスフェクション * 24時間50から70パーセント翌日のコンフルエントのため10 cmディッシュにトランスフェクション、プレート2.5 × 10 6個の細胞の前に。 <ol><li>一つ後のウイルスを下すために、単一の細胞をトランスフェクトするとなるとDNAを調製してこのプロトコルを開始します。最初に、FuGENE 6トランスフェクション試薬ロシュ、細胞がDNAを取るように脂質の試薬を希釈する。 1.5 mlチューブに、ピペット30μlのFuGENE 6トランス600ulの無血清培地に（SFDMEM）。それは媒体に配信されるようにトランスフェは、チューブの側面に接触させてように注意してください。このトランスフェと5分間室温でSFDMEMミックスとインキュベートしてみましょう。 </li><li>チューブのキャップでは、それぞれの第3世代ウイルスパッケージングベクトル（PMDL、pRSVとpVSV - G）は、4μgのレンチウイルスプラスミド4μgのを混ぜる。レンチウイルスプラスミドは、パッケージングベクターがレンチウイルスを作るために必要なすべての他のタンパク質の遺伝子を含んでいる間、ウイルスは、それが感染するすべての細胞のゲノムに挿入するDNAが含まれています。 </li><li>チューブを反転させて蓋をし、混合数回を閉じます。 </li><li>室温で15-20分インキュベートする。 </li><li> 293プレートへのチューブのピペット全体の内容。前後に傾斜板で静かに混和。 </li><li>インキュベーターにセルを戻すと、ウイルス産生が収穫前48〜96時間継続することができます。 </li><li>インキュベーションの間、293細胞は、レンチウイルスコンストラクトのRNAのコピーを作成し、レンチウイルスプラスミドから目的のDNAを転写する。彼らはまた、ウイルス蛋白質へのパッケージングベクター中の遺伝子を転写し、翻訳する。これらのタンパク質は、ウイルスが転写するを逆転させ、それが感染するすべての細胞のゲノムに挿入するあなたのレンチウイルスコンストラクトのRNAのバージョンを含むウイルスを作るに進んでください。 </li></ol>第2部：レンチウイルス収穫<ol><li>潜伏期間の後、インキュベーターから細胞を取り出し、現在ウイルスを含む上清を除去するために使い捨て、10mlのシリンジを使用してください。上清10mlを約あるでしょう。 </li><li>先端に0.45μMシリンジフィルターを取り付け、ベックマンの超遠心管にウイルスをフィルタリングする。フィルタはウイルスではなく、細胞が通過することができます。 </li><li>廃棄する前に、すべてのウイルス産生細胞、培養プレート、45分間10％の漂白剤のシリンジとフィルターをインキュベートする。 </li></ol>パート3：超遠心法を経由してレンチウイルス濃度<ol><li> SW - 41TiまたはSW - 28ローターバケットに遠心管をロードする。 </li><li>すべてのウイルスを含むチューブに0.2グラム内のバランスをとるために無血清DMEMを使用してください。 </li><li>超遠心機内部のローターを配置する前に、ローターバケットをシャットダウンして、ローターの上にそれらをフック封印</li><li> 4℃で超遠心機で90分間ウイルスをスピン· SW - 41Tiローターで25000回転またはSW - 28ローターで24000 rpmでC。 </li><li>組織培養フードの中で、10％の漂白剤を入れた容器に上清を注ぐために逆さにチューブを反転させる。 </li><li>ペレットの周りに余分な液体を吸収し、5分を超えないために便利なラックにチューブを反転させるためにキムワイプを使用してください。 </li><li>ウイルスのペレットを再度中断するには、約20回上下にピペッティングして、チューブに100μLの滅菌PBSを追加するには、フィルターチップを使用。 </li><li>再懸濁完了する℃で一晩4℃でウイルスを残す。一つは、約1週間と1年まで-80℃で4℃でウイルスプレップを保存することができます。廃棄するまでに10％漂白剤ですべての空のチューブを扱うことをお忘れなく。 </li><li>いずれかの1週間よりも長くウイルスprepを使用しようとする場合、最小限の将来の実験のための5 -20μlののアリコートと滴定のための5μlの3の1アリコートを行います。マイナス80℃アリコートを凍結して保存する。 </li></ol>パート4：フローサイトメトリーを用いてレンチウイルス滴定<ol><li>次のステップは、フローサイトメトリーにより、ウイルス力価を決定することです。このメソッドは、レンチウイルスプラスミドは、蛍光レポーター遺伝子が含まれている場合に動作します。アイデアは、ウイルスが細胞に感染した場合、それはその細胞のゲノムにレンチウイルスプラスミドDNAを導入することです。感染細胞は、転写と翻訳が含まれている遺伝子をレンチウイルスプラスミドにし、蛍光レポーター遺伝子が含まれていると、感染細胞は蛍光を発するでしょう。蛍光のレベルは、ウイルスの効力、または力価を、表します。 </li><li>滴定する各ウイルス株については、完全なメディアを1.5 ml（DMEM、ペン連鎖球菌、グルタミン、FBS）に濃縮されたウイルスの3μlを希釈する。 </li><li> 96ウェルプレートの1行目の井戸1-6に、以下の連続希釈を行います：全6ウェルに完全DMEM培地を100μlを加える。ウェルを100μlを加えるだけでなく秒に、（これは未染色のコントロールです）がないウイルスとメディアの100μLを追加する第三者に、希釈したウイルス液100μlを（希釈ウイルスと同等の最高1ミリリットルである）最初に追加するによく秒からミックス、よくよく第五に、よく3日から100 mlを加えるだけでなく第四井戸から、目と6番目に100μLを加えるも5日から100μlを加える4位に。最後に第六井戸から100ulを取り出して、漂白剤で捨てる。各ウェルは、その前のに比べてウイルスの半分のボリュームを持っている場所連続希釈を行った。 </li><li> 200μlのためにすべてのウェルの総量をもたらす。これらが最も集中してウイルスを力価測定のために働く必要は推奨希釈率、であることに注意してください。ウイルスは、力価の低い場合、または未濃縮場合、1つは、希釈を調整する必要があります。 </li><li>バイオハザード廃棄物容器に廃棄する前に45分間、10％漂白剤で使用されていない希釈したウイルスを扱う。 </li><li> 6ウェルの各々に〜15,000健全な、活発に分裂した293細胞を追加。シングル96ウェルプレートは、16のウイルスプレップを力価に使用することができます。 </li><li> 5％のCOと37度のインキュベーターにセルを返す2、感染が少なくとも48時間続行することができます。 </li><li>インキュベーション後、細胞をフローサイトメーターで（我々の場合の蛍光で）レポーターの発現を分析している。ウイルス粒子は、ウェルあたりの蛍光細胞の割合によって定量される。 </li></ol>次の式でmlあたり感染単位数を計算します。 <div style="text-align:center;"> （＃感染Xのレポーター陽性細胞の日にウェルあたりの細胞の）X 1000 ______________________________________________________ ベクトルのmlのウェルに添加 </div>パート5：代表的な結果： 48時間のインキュベーション期間の後のトランスフェクションした後、293細胞のプレートは、蛍光灯約90％になっているはず。蛍光細胞の割合が高いウイルス産生細胞の割合が高いの指標です。 再懸濁するときに遠心分離した後、ウイル​​スペレットはほとんど見えないです。ペレットは透明で非常に小さいため、これは正常です。 細胞は細胞ごとに1つしかウイルスの統合を受けている（蛍光灯）に感染したときに一つだけ正確な力価の計算を達成することができるフローサイトメーターの結果の解釈、注意してください。これが事実であることを保証するために、計算のための&lt;15％の感染率を持つ細胞を使用してください。高い感染率を有する細胞は、おそらく、セルごとに複数のウイルスintegrantsを受けている。手順2で提案された希釈液は、感染症のこの範囲内でいくつかのサンプルを生成する必要があります。理想的には、最後の力価を計算することができますが、必要に応じて、単一のサンプルを用いて力価を計算できるから滴定プロット（直線）を生成するためにいくつかのサンプルを使用してください。一つは、1.0 × 10 7 TU非濃縮したウイルスのための/ mlおよび1.0 × 10 8濃縮したウイルスのためのTU / mlの力価が期待できます。

<ol>
	<li>Reiser, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+concentration+of+pseudotyped+HIV-1-based+gene+transfer+vectors..">Production and concentration of pseudotyped HIV-1-based gene transfer vectors..</a> Gene Therapy. , 72-7211 (2000).</li><li>Dull, T., Zufferey, R., Kelly, M., Mandel, R. J., Nguyen, M., Trono, D., Naldini, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> J Virol. 72 (11), 8463-8471 (1998).</li></ol>

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>DMEM HEK 293T cells Penicillin-streptomycin Glutamine Phosphate-buffered saline FBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>FuGENE 6 transfection reagent pSico mCherry pMDL pRSV pVSVG</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Bleach</td>
<td>&nbsp;</td>
<td>Clorox</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10-cm cell culture dishes 96 well cell culture dishes </td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>353003</td>
<td>&nbsp;</td>
<tr>
<td>Multichannel pipette</td>
<td>&nbsp;</td>
<td>Rainin</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Filtered pipette tips</td>
<td>&nbsp;</td>
<td>Rainin</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1.5ml Eppendorf tubes Syringe Syinge filter (0.45-&mu;m)</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Ultracentrifuge tubes</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Tissue culture incubator at 5% CO2</td>
<td>&nbsp;</td>
<td>Coulter</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Beckman SW41T.I rotor</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Beckman ultracentrifuge</td>
<td>&nbsp;</td>
<td>Beckman </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescent microscope</td>
<td>&nbsp;</td>
<td>Coulter</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>FACS Array</td>
<td>&nbsp;</td>
<td>Beckman</td>
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lentivirus production

RNA干渉（RNAi）は、生きた細胞内の遺伝子サイレンシングのシステムです。 RNAi実験では、短い干渉RNA（siRNA）のシーケンスの相同遺伝子は、転写後の状態で沈黙している。ショートヘアピンRNA、siRNAに前駆体は、種々の細胞型におけるRNAiを可能に、レンチウイルスを用いて表現することができる。このようなヒト免疫不全ウイルスのようなレンチウイルスは、感染の両方で割ると、非分裂細胞に対応しています。我々は、レンチウイルスをパッケージ化する手順を説明します。パッケージングは​​、DNAベクターから有能なウイルスの準備を指します。レンチウイルスベクターの生産システムは、天然のウイルスゲノムは、個々のヘルパープラスミドコンストラクトに分割された&quot;分割&quot;システムに基づいています。 4つの別々のベクターに異なるウイルスの要素のこの分裂は、レンチウイルスゲノムの不定組換えによる複製が可能なウイルスを作成することのリスクを減少させる。ここでは、関心と3種類のパッケージでベクトル（P - VSVG、pRSV、PMDL）のshRNAを含むベクターを、一過性のヒト293細胞にトランスフェクトされています。少なくとも48時間のインキュベーション期間の後、ウイル​​スを含む上清を回収し、濃縮する。最後に、ウイルスの力価は、レポーター（蛍光灯）フローサイトメーターによる式によって決定されます。

Watch this Scientific Journal Video about Lentivirus Production at JoVE.com

Lentivirus Production

レンチウイルスを作成するには、DNAベクターは、ヒト293細胞にトランスフェクトされています。収穫後、上清を濃縮し、ウイルス力価は、フローサイトメーターと蛍光式によって決定されます。

レンチウイルスの生産

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are ...

Research

JoVE Journal

Biology

65.5K ビュー.  University of California, San Francisco - UCSF. レンチウイルスを作成するには、DNAベクターは、ヒト293細胞にトランスフェクトされています。収穫後、上清を濃縮し、ウイルス力価は、フローサイトメーターと蛍光式によって決定されます。

ビデオ: Lentivirus Production

Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1. Recent advances in this technology have enabled high resolution comparison of whole genomes for the identification of genetic alterations in cancer and other genetic diseases 2. The Sub-Megabase Resolution Tiling-set array (or SMRT) array is comprised of a set of approximately thirty thousand overlapping bacterial artificial chromosome (BAC) clones that span the human genome in ~100 kilobase pair (kb) segments 2. These BAC targets are individually synthesized and spotted in duplicate on a single glass slide 2-4. Array CGH is based on the principle of competitive hybridization. Sample and reference DNA are differentially labeled with Cyanine-3 and Cyanine-5 fluorescent dyes, and co-hybridized to the array. After an incubation period the unbound samples are washed from the slide and the array is imaged. A freely available custom software package called SeeGH (www.flintbox.ca) is used to process the large volume of data collected - a single experiment generates 53,892 data points. SeeGH visualizes the log2 signal intensity ratio between the 2 samples at each BAC target which is vertically aligned with chromosomal position 5,6. The SMRT array can detect alterations as small as 50 kb in size 7. The SMRT array can detect a variety of DNA rearrangement events including DNA gains, losses, amplifications and homozygous deletions. A unique advantage of the SMRT array is that one can use DNA isolated from formalin fixed paraffin embedded samples. When combined with the low input requirements of unamplified DNA (25-100ng) this allows profiling of precious samples such as those produced by microdissection 7,8. This is attributed to the large size of each BAC hybridization target that allows the binding of sufficient labeled samples to produce signals for detection. Another advantage of this platform is the tolerance of tissue heterogeneity, decreasing the need for tedious tissue microdissection 8. This video protocol is a step-by-step tutorial from labeling the input DNA through to signal acquisition for the whole genome tiling path SMRT array.

This video is a technical demonstration of the hybridization protocol for whole genome tiling path array CGH, which scans the entire human genome using only 25-100 ng of DNA that can be isolated from a variety of sources, including archival formalin fixed material.

全ゲノムアレイ比較ゲノムハイブリダイゼーションの技術デモンストレーション

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1.  Recent ...

生物学

Large Insert Environmental Genomic Library Production

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approaches have emerged, enabling researchers to bridge this cultivation gap by capturing the genetic content of indigenous microbial communities directly from the environment. To this end, genomic DNA libraries are constructed using standard albeit artful laboratory cloning techniques. Here we describe the construction of a large insert environmental genomic fosmid library with DNA derived from the vertical depth continuum of a seasonally hypoxic fjord. This protocol is directly linked to a series of connected protocols including coastal marine water sampling [1], large volume filtration of microbial biomass [2] and a DNA extraction and purification protocol [3]. At the outset, high quality genomic DNA is end-repaired with the creation of 5 -phosphorylated blunt ends. End-repaired DNA is subjected to pulsed-field gel electrophoresis (PFGE) for size selection and gel extraction is performed to recover DNA fragments between 30 and 60 thousand base pairs (Kb) in length. Size selected DNA is purified away from the PFGE gel matrix and ligated to the phosphatase-treated blunt-end fosmid CopyControl vector pCC1 (EPICENTRE <a href="http://www.epibio.com/item.asp?ID=385">http://www.epibio.com/item.asp?ID=385</a>). Linear concatemers of pCC1 and insert DNA are subsequently headfull packaged into phage particles by lambda terminase, with subsequent infection of phage-resistant E. coli cells. Successfully transduced clones are recovered on LB agar plates under antibiotic selection and archived in 384-well plate format using an automated colony picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the CopyControl Fosmid Library Production Kit from EPICENTRE and the published works of multiple research groups [4-7]. Each step is presented with best practice in mind. Whenever possible we highlight subtleties in execution to improve overall quality and efficiency of library production. The whole process of fosmid library production and automated colony picking takes at least 7-10 days as there are many incubation steps included. However, there are several stopping points possible which are mentioned within the protocol.

Construction of a fosmid library with environmental genomic DNA isolated from the vertical depth continuum of a seasonally hypoxic fjord is described. The resulting clone library is picked into 384-well plates and archived for downstream sequencing and functional screening by the application of an automated colony picking system.

大規模な挿入環境ゲノムライブラリーの製造

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent ...

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing the pluripotent state in mouse fibroblasts.1 The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.2 Additionally, James Thomson et al. reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.3
iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells.
Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.

We demonstrate the protocol for the generation of induced pluripotent stem cells from human somatic cells using lentivirus-mediated delivery of the human factors Oct4, Sox2, Nanog, and Lin28. Pluripotency was confirmed by morphology and the presence of embryonic stem (ES) cell-specific markers.

StemgentヒトTFのレンチセットでヒト線維芽細胞を再プログラムすることによって誘導多能性幹細胞の生成

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing ...

A Protocol for the Production of KLRG1 Tetramer

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. KLRG1 exists both as a monomer and as a disulfide-linked homodimer. This well-conserved receptor is found on the most mature and recently activated NK cells as well as on a subset of effector/memory T cells.
Using KLRG1 tetramer as well as other methods, E-, N-, and R-cadherins were identified as KLRG1 ligands. These Ca2+-dependent cell-cell adhesion molecules comprises of an extracellular domain containing five cadherin repeats responsible for cell-cell interactions, a transmembrane domain and a cytoplasmic domain that is linked to the actin cytoskeleton. 
Generation of the KLRG1 tetramer was essential to the identification of the KLRG1 ligands. KLRG1 tetramer is also a unique tool to elucidate the roles cadherin and KLRG1 play in regulating the immune response and tissue integrity.

This protocol describes the production of KLRG1 tetramer, which is a powerful tool for the analysis of KLRG1 ligands.

KLRG1テトラマーの生産のためのプロトコル

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

遺伝学的およびエピジェネティクス解析のためのパラフィン包埋材料からのDNA抽出

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for laboratory personnel. Pantropic lentiviruses, such as the commonly used VSV-G pseudotype, are a valuable tool for studying gene function because they can transduce many cell types, including non-dividing cells. However, researchers may wish to avoid production and centrifugation of pantropic viruses encoding oncogenes due to higher biosafety level handling requirements and safety issues. Several potent oncogenes, including c-Myc and SV40 large T antigen, are known to enhance production of induced pluripotent stem cells (iPSC). All other known iPSC-inducing genetic changes (OCT4, SOX2, KLF4, NANOG, LIN28, and p53 loss of function) also have links to cancer, making them of relatively high safety concern as well. 
While these cancer-related viruses are useful in studying cellular reprogramming and pluripotency, they must be used safely. To address these biosafety issues, we demonstrate a method for transduction of human cells with ecotropic lentivirus, with additional emphasis on reduced cost and convenient handling. We have produced ecotropic lentivirus with sufficiently high titer to transduce greater than 90% of receptor-expressing human cells exposed to the virus, validating the efficacy of this approach. 
Lentivirus is often concentrated by ultracentrifugation; however, this process takes several hours and can produce aerosols infectious to human biomedical researchers. As an alternative, viral particles can be more safely sedimented onto cells by complexation with chondroitin sulfate and polybrene (CS/PB). This technique increases the functional viral titer up to 3-fold in cells stably expressing murine retrovirus receptor, with negligible added time and cost. Transduction of human dermal fibroblasts (HDFs) is maximally enhanced using CS/PB concentrations approximately 4-fold lower than the optimal value previously reported for cancer cell lines, suggesting that polymer concentration should be titrated for the target cell type of interest. We therefore describe the use of methylthiazolyldiphenyl-tetrazolium bromide (MTT) to assay for polymer toxicity in a new cell type. We observe equivalent viability of HDFs after viral transduction using either polymer complexation or the standard dose of polybrene (PB, 6 &mu;g/ml), indicating minimal acute toxicity.
In this protocol, we describe the use of ecotropic lentivirus for overexpression of oncogenes in human cells, reducing biosafety risks and increasing the transduction rate. We also demonstrate the use of polymer complexation to enhance transduction while avoiding aerosol-forming centrifugation of viral particles.

Lentiviruses are a valuable research tool for exploring gene function; however, researchers may wish to avoid production of pantropic lentivirus encoding known or suspected oncogenes. As an alternative, we present a safer protocol for use of ecotropic lentivirus on human cells modified to express the ecotropic receptor mSlc7a1.

強化されたバイオセーフティのためのポリマー複合エコトロピックレンチウイルスによるヒト細胞のトランスダクション

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

EMBLでMultiBacタンパク質複合体生産プラットフォーム

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity &#8211; a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish is a powerful model system for developmental biology and human disease research due to their genetic similarity with higher vertebrates. This protocol describes a methodology to create haploid zebrafish embryos that can be utilized for forward screen strategies to identify recessive mutations in genes essential for early embryogenesis.

半数ゼブラフィッシュ胚の生成により<em&gt;体外</em&gt;受精

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜タンパク質の緑色蛍光タンパク質ベースの発現スクリーニングで<em&gt;エシェリヒア·コリ</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Production of Double-stranded DNA Ministrings

We constructed linear covalently closed (LCC) DNA minivectors as a non-viral gene-delivery vector alternative produced via a simple platform in vivo. DNA ministrings possess a heightened safety profile and also efficiently deliver DNA cargo to targeted cells. Conventional DNA vectors carry undesirable prokaryotic sequences, including antibiotic resistance genes, CpG motifs, and bacterial origins of replication, which may lead to the stimulation of host immunological responses. The bioavailability of conventional DNA vectors is also compromised due to their larger molecular size. Their circular nature may also impart chromosomal integration, leading to insertional mutagenesis.
Bacterial sequences are excised from DNA minivectors, leaving only the gene of interest (GOI) and necessary eukaryotic expression elements. Our LCC DNA minivectors, or DNA ministrings, are devoid of immunogenic bacterial sequences; therefore improving their bioavailability and GOI expression. In the event of vector integration into the chromosome, the LCC DNA ministring will lethally disrupt the host chromosome, thereby removing the potentially dangerous mutant from the proliferating cell population. Consequently, DNA ministrings offer the benefits of &#39;minicircle&#39; DNA while eliminating the potential for undesirable vector integration events. In comparison to conventional plasmids and their isogenic circular covalently closed (CCC) counterparts, DNA ministrings demonstrate superior bioavailability, transfection efficiency, and cytoplasmic kinetics - they thus require lower amounts of cationic surfactants for effective transfection of target cells.
We have constructed a one-step inducible in vivo system for the production of DNA ministrings in Escherichia coli that is simple to use, rapid, and scalable.

The linear covalently closed (LCC) DNA minivector (DNA ministring) is a non-viral gene delivery vector offering high transfection efficiency and is relevant to any DNA delivery application. The production system is simple, rapid, and scalable in vivo. The following protocol provides visual step-by-step instructions for the production of DNA ministrings.

二本鎖DNA Ministringsの生産

We constructed linear covalently closed (LCC) DNA minivectors as a non-viral gene-delivery vector alternative produced via a simple platform in vivo. DNA ...