Name: selection-dependent and independent generation of crispr/cas9-mediated gene knockouts in mammalian cells
Uploaded: 2017-06-16T15:00:00
Description: Watch this Scientific Journal Video about Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells at JoVE.com

著者は、実験支援のためにGissell Sanchez、Megan Lee、Jason Estep、試薬を共有するWeifeng GuとXuemei Chenを認めたいと思っています。

1.所望の欠失周辺の相同性領域の同定注：選択ベース編集を使用している場合のみ必要です。 <ol><li> HDR鋳型（ 図1A ）の相同性アームとして機能する所望の欠失座の両側に、最初は1.5-2kbの2つの領域を選択する。クローニングを容易にするために、いずれかの鎖（GGTCTC）上のBsaI認識部位を欠く領域を同定する。 BsaI部位が避けられない場合は、別の...

<ol>
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CRISPR / Cas9システムは、安定したゲノム改変の効率的な生成を可能にし、他の一時的な操作方法のより一貫した代替物を提供する。ここでは、哺乳動物細胞株におけるCRISPR / Cas9遺伝子ノックアウトの迅速な同定のための2つの方法を提示した。どちらの方法も細胞材料をほとんど必要としないため、クローン培養の初期段階で試験を行い、時間と試薬を節約できます。両方の方法の効率を上げ...

<html>安定した遺伝子改変は、細胞摂動の一時的な方法よりも利点を提供し、その効率および持続時間は可変であり得る。ゲノム編集は、亜鉛フィンガーヌクレアーゼ1,2,3,4,5、転写アクチベーター様エフェクターヌクレアーゼ（TALEN）6,7,8,9などの標的特異的ヌクレアーゼの開発のために近年ますます一般的になってきているクラスター化され、規則的に間隔を置いて配置された短いパリンドローム反復（CRISPR）システム10に由来するRNA誘導ヌクレアーゼを含む。 CRISPR / Cas9編集機は、細菌や古細菌がウイルス感染を防御するために使用する免疫システムに適合していますass = &quot;xref&quot;&gt; 11,12,13。このプロセスでは、侵入するウイルス配列の短い20〜30ntフラグメントが、繰り返しユニット14,15に隣接する「スペーサー」としてゲノム座に組み込まれる。その後の転写およびRNAプロセシングにより、トランス活性化crRNA 17 （tracrRNA）とともに、エフェクターCas9エンドヌクレアーゼと組み立てられる小さなCRISPR関連RNA16（crRNA）が生成される。このようにして、crRNAは、相補性ウイルスDNA配列を切断し、さらなる感染を予防するために、複合体を誘導するCas9標的化への特異性を提供する18,19。標的DNA中のいずれかの「プロトスペーサー」配列は、短いprotospacer隣接モチーフ（PAM）、ストレプトコッカス・ピオゲネス Cas9の場合のNGG <sup class = &quot;xref&quot;&gt; 20。宿主のCRISPR遺伝子座のスペーサーの近くにPAM配列がないことは、自己と非自己とを区別し、宿主の標的化を妨げる。その普遍性と柔軟性のために、この生物学的システムは、ほぼ全てのPAMに隣接するDNA部位を標的とすることができるように、ゲノム編集に強力に適合している。このバージョンでは、さらなる改変により、crRNAおよびtracrRNAが、Cas9タンパク質21にロードされる単一のガイドRNA（sgRNA）成分に融合した。 Cas9およびsgRNAを真核細胞で発現させると、Cas9タンパク質は、標的遺伝子座で両方のDNA鎖を切断する。相同配列の適切な領域が存在しない場合、細胞は、典型的には小さな欠失またはまれに挿入を導入する非相同末端結合（NHEJ） 22,23,24を介してこのブレークを固定する。オープンをターゲットとする場合修復は、おそらく非機能的なタンパク質産物を産生する翻訳フレームシフトにつながる。対照的に、大きな相同性領域を有する外因性鋳型を提供する場合、細胞は相同性指向修復により二本鎖切断を固定することができる25,26 。この経路は、切除可能な選択マーカー27の導入と相まって、ゲノムにおけるより大きな正確な欠失、置換または挿入を可能にする。 ここでは、これらの2つのCRISPR / Cas9法（ 図1A ）のいずれかによってノックアウト細胞株を生成するためのプロトコールを提示する。 NHEJアプローチは、単一のsgRNA切断部位および選択非依存性スクリーニングを使用するため、ほとんど準備を必要としない。この方法を使用する場合、ノックアウトを生成する可能性が最も高い、転写物の5 &#39;末端近くのエクソンに相補的な誘導RNAを設計しなければならない。変更後この場合、ゲノムへのイオンの量は少なく、ノックアウトクローンのスクリーニングはドットブロットに基づいており、タンパク質産物はハイスループットの様式で評価される。我々はELAV様1タンパク質（ELAVL1）ノックアウト系の生成を例として使用する。第2のアプローチは相同性指向修復（HDR）に依拠し、数十kbの欠失を可能にする目的の遺伝子または関心領域にまたがる2つのsgRNA切断部位を使用する。切断部位に隣接する2つの相同性領域を有するプラスミドは、ノックアウト生成の効率を増加させる選択可能な耐性マーカーを導入する置換テンプレート（ 図1B ）を提供する。この方法は、適切に設計された相同性アームを用いて遺伝子改変を導入するために適合させることもできる。この場合、新しいDNA断片の組み込みにより、PCRベースのスクリーニングが可能になる（ 図1C ）。ここでは、Pumilio RNA binding family member 2（PUM2）ノックアウト系統の生成を例として使用します。

<table border="0" cellpadding="0" cellspacing="0" width="740">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">Competent E. coli cells</td>
			<td style="width:95px;"></td>
			<td style="width:120px;"></td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">plasmid prep kit</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pSpCas9(BB) plasmid</td>
			<td>Addgene</td>
			<td>42230</td>
			<td>Ran et al 2013, cloning sgRNAs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BbsI enzyme</td>
			<td>ThermoFisher</td>
			<td>FD1014</td>
			<td>Ran et al 2013, cloning sgRNAs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">T7 DNA ligase</td>
			<td>NEB</td>
			<td>M0318L</td>
			<td>Ran et al 2013, cloning sgRNAs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tango Buffer</td>
			<td>ThermoFisher</td>
			<td>BY5</td>
			<td>Ran et al 2013, cloning sgRNAs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PlasmidSafe exonuclease</td>
			<td>Epicentre</td>
			<td>E3105K</td>
			<td>Ran et al 2013, cloning sgRNAs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Q5 hot start high fidelity polymerase</td>
			<td>NEB</td>
			<td>M0494A</td>
			<td>HA amplification</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pUC19-BsaI</td>
			<td></td>
			<td></td>
			<td>Modified pUC19 plasmid, mutated existing BsaI site and inserted two outward facing BsaI sites after BamHI/EcoRI digestion</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pGolden-Neo</td>
			<td>Addgene</td>
			<td>51422</td>
			<td>Resistance cassette</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pGolden-Hygro</td>
			<td>Addgene</td>
			<td>51423</td>
			<td>Resistance cassette</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BsaI enzyme</td>
			<td>NEB</td>
			<td>R3535S</td>
			<td>Homology arm contruction</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">T7 DNA ligase</td>
			<td>NEB</td>
			<td>M0318L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PlasmidSafe exonuclease</td>
			<td>Epicentre</td>
			<td>E3105K</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Toothpicks</td>
			<td></td>
			<td></td>
			<td>bacterial PCR</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Taq polymerase</td>
			<td></td>
			<td></td>
			<td>bacterial colony PCR</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEK293 human cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS</td>
			<td>Corning</td>
			<td>MT35010CV</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-Strep (opt.)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6 well plates</td>
			<td>BioLite</td>
			<td>12556004</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TransIT-LTI Transfection Reagent</td>
			<td>Mirus</td>
			<td>MIR2300</td>
			<td>for lipofection only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Opti-MEM</td>
			<td>ThermoFisher</td>
			<td>31985062</td>
			<td>for lipofection only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">G418 (Neomycin)</td>
			<td>Sigma Aldrich</td>
			<td>A1720-5G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hygromycin</td>
			<td>Sigma Aldrich</td>
			<td>H3274-250MG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 well plates</td>
			<td>ThermoFisher</td>
			<td>12556008</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">Passive Lysis Buffer, 5x</td>
			<td>Promega</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">1xSDS loading buffer</td>
			<td></td>
			<td></td>
			<td>recipe decribed in protocol</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nitrocellulose Membrane</td>
			<td>Bio-Rad</td>
			<td>162-0115</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TBST</td>
			<td></td>
			<td></td>
			<td>recipe decribed in protocol</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dehydrated milk</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SuperSignal West Dura Extended Duration Substrate</td>
			<td>ThermoFisher</td>
			<td>34075</td>
			<td>for HRP-conjugated secondary antibodies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Extracta DNA prep for PCR</td>
			<td>Quantabio</td>
			<td>95091-025</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KOD polymerase</td>
			<td>Novagen</td>
			<td>71316</td>
			<td></td>
		</tr>
	</tbody>
</table>

selection-dependent and independent generation of crispr/cas9-mediated gene knockouts in mammalian cells

CRISPR / Cas9ゲノムエンジニアリングシステムは、わずかな労力で正確なゲノム編集を可能にすることによって生物学に革命をもたらしました。特異性を与える単一のガイドRNA（sgRNA）によって導かれ、Cas9タンパク質は、標的遺伝子座で両方のDNA鎖を切断する。 DNA破壊は、非相同末端結合（NHEJ）または相同性指向修復（HDR）のいずれかを引き起こす可能性がある。 NHEJは、フレームシフト突然変異をもたらす小さな欠失または挿入を導入することができ、一方、HDRはより大きくより正確な摂動を可能にする。ここでは、確立されたCRISPR / Cas9法を下流の選択/スクリーニングの2つの選択肢と結びつけることによってノックアウト細胞株を生成するためのプロトコールを提示する。 NHEJアプローチでは、単一のsgRNA切断部位および選択非依存性スクリーニングを使用し、ここでタンパク質産生は、ハイスループット様式でドットイムノブロットによって評価される。 HDRアプローチは、目的の遺伝子にまたがる2つのsgRNA切断部位を使用する。提供されたHDRテンプレートと一緒に、この方法は削除を達成することができます挿入された選択可能な抵抗マーカーによって助けられた数十kbの大きさであった。それぞれの方法の適切なアプリケーションと利点について説明します。

ELAVL1ノックアウト系統の作製には、頑強な抗体が利用できたので、単一のsgRNA（ 図1A 、左）を用いた編集を行い、次にドットイムノブロットを行った。 3つのsgRNAを独立してトランスフェクトして、効率を比較し、得られたクローンの標的効果を排除した。クローン集団からの細胞溶解物を2つのニトロセルロースメンブレン上に集め、ブロット?...

Watch this Scientific Journal Video about Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells at JoVE.com

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

体細胞株を遺伝子操作する能力の最近の進歩は、基礎研究および応用研究の大きな可能性を秘めている。ここでは、選択マーカーを使用した場合と使用しない場合の、哺乳類細胞株におけるCRISP / Cas9生成ノックアウト産生およびスクリーニングのための2つのアプローチを提示する。

哺乳類細胞におけるCRISPR / Cas9媒介遺伝子ノックアウトの選択依存性および独立した世代

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide ...

The goal of this procedure is to isolate and identify clonal knockout cell lines generated by two types of CRIPSR/Cas9-mediated genome editing. One method detects protein knockout caused by nonhomologous end joining while the other detects genomic perturbations generated by homology-directed repair. This method is particularly useful in the field of molecular biology, where generation of cell lines with genetic perturbations is an important tool in dissecting gene function.The main advantage of this technique is that it is a quick and relatively high-throughput method that can be easily executed using basic molecular biology techniques. Nonhomologous end joining introduces small deletions or insertions, and are assessed by a dot immunoblot, while homology-directed repair allows for larger and more precise perturbations, and are assessed by colony PCR. This experiment uses T-REx-293 cells cultured in DMEM media, supplemented with 10%FBS at 37 degrees Celsius, and 5%carbon dioxide.Prior to transvection, plate the cells onto six well plates and include a well for an untransvected control. Grow the cells to approximately 70%confluency. Transvect the cells with 2.5 micrograms of total plasmid using a commercial transvection protocol.For transvection of cells without selection, use 2.5 micrograms of Cas9-sgRNA plasmid. For transvection of cells with selection, use 0.75 micrograms of Cas9-sgRNA1, 0.75 micrograms of Cas9-sgRNA2, and one microgram of homologous recombination template. Incubate the transvected cells and the untransvected control at 37 degrees Celsius and 5%carbon dioxide for 48 hours.For cells transvected with selection, begin the drug selection by treating the cells with the appropriate drug, which is Neomycin in this case. Return the six-well plate to the incubator. Observe the treated cells once a day to check for rates of cell death, until all cells in the untransvected control die.To isolate clonal populations, first grow the cells to 100%confluency in the original well after selection. Next make serial dilutions of the cells and count the cells. Since the proper generation of monoclonal colonies is key to obtaining a full knockout, care must be taken to determine the correct dilution for seating cells at the desired density.Once the correct dilution has been determined, seat the cells into 96-well plates at a density of 0.33 cells per well. Seating three 96=well plates is a good starting point to ensure isolation of more than one correct clone. Over a two-to-four week period, observe the colonies until colonies are visible to the eye.These representative images show colonies at different stages of growth. A single cell at seating, cells at day five, and a well-established colony at day 10. Using a sterile pipette tip, pick visible colonies with care and precision, and reseat in new wells to encourage monolayer growth.Continue growing the cells at 37 degrees Celsius and 5%carbon dioxide. Knockout candidates without selection are screened by dot immunoblot to assess the protein product in a high-throughput manner. After growing individual clones to 50 to 100%confluency, dislodge the monolayer of cells by pipetting within the well.Aliquot 90 microliters of the 100 microliter total volume from each well to a clean microcentrifuge tube. For the purposes of this video, only two samples will be processed. Spin down at 6, 000 RPM for five minutes, remove the media, and lyse the cell pallet in 10 microliters of 1X SDS loading buffer.Add 90 microliters of new media to the remainder of the cells in the well, and return the plate to the incubator to continue propagating the cells. Pipette one microliter of cell lysate onto a dry nitrocellulose membrane to form a dot. Blot each sample twice on two separate membranes, creating two identical patterns of samples.Block the membranes in 5%milk in TBST at room temperature, with rocking, for one hour. After one hour, blot the membranes with primary antibodies at the recommended primary antibody dilution, and TBST plus 5%milk. On one membrane use the primary antibody against the target protein, ELAVL1 in this experiment.On the other membrane, use the primary antibody for a control protein that is not expected to change, PUM2 is used here. Incubate at room temperature for one hour. Remove the primary antibody solution from each blot, add TBST, and incubate for five minutes.In this manner, wash the blots three times with TBST. Next, add to each membrane the appropriate HRP conjugated secondary antibody, and blot at room temperature for one hour. After one hour, wash the membranes three times with TBST for five minutes each time.Apply a chemiluminescent substrate solution to the blots, following the manufacturer's instructions, and image the blotted membranes on a digital chemiluminescence imager. Lastly, quantify dot intensities for the target and control proteins using the appropriate software, and analyze the results as described in the text protocol. Candidates with the selectable resistance marker are screened by colony PCR.Begin this procedure by duplicating individual colonies in a new 96-well plate, and growing them to 100%confluency for preparation of cell lysates. Remove the media from one set of the clones, resuspend the cells from each well in 30 microliters of extraction buffer from a commercial DNA preparation kit, and transfer to a clean 1.5 milliliter microcentrifuge tube. Heat the solution to 96 degrees Celsius for 15 minutes, and then let cool to room temperature.Add 30 microliters of stabilization buffer to each microcentrifuge tube and mix well. To identify wild-type and monoallelic or biallelic mutant lines by colony PCR, design two separate sets of PCR primers to amplify regions around the homology arms based on either successful or unsuccessful integration of the resistance cassette. On each side, use a common primer that anneals outside of the homology arm.To test for the wild-type allele, use the common primer with the corresponding paired primer that is complementary to the endogenous sequence spanning the homology region. To test for the desired mutation, use the common primer with another paired primer, complementary to the inserted resistance cassette. For each sample to be tested, prepare a reaction mixture containing the following, 0.5 microliters of cell lysate, 1.25 microliters of 10X KOD buffer, 0.75 microliters of 25 millimolar magnesium sulfate, 1.25 microliters of two millimolar dNTPs, 0.375 microliters of forward primer, 0.375 microliters of reverse primer, 0.25 microliters of KOD polymerase, and 7.75 microliters of water.Perform PCR using the following conditions, 95 degrees Celsius for two minutes, followed by 25 cycles of 95 degree Celsius for 20 seconds, primer melting temperature for 10 seconds, and 70 degrees Celsius for 20 seconds per KB.Subsequently, visualize the PCR products by agarose gel electrophoresis. Genome editing using non-homologous end joining was performed to generate ELAVL1 knockout lines followed by dot immunoblot, probing for ELAVL1 and PUM2. Clones that displayed little control signal were marked with an X, and excluded from further analysis.Clones with low ELAVL1 signals were further tested by western blot. Confirmed candidates are indicated in green, and invalidated candidates are indicated in gray. This graphic shows the ratios of ELAVL1 to control protein signal in increasing order.In most cases, clonal populations with the lowest relative ELAVL1 signal were correctly identified as knockouts. Candidates generated by the homology directed repair method were screened by colony PCR, and the PCR products visualized by agarose gel electrophoresis. Shown are example results using primers spanning the upstream integration junction using endogenous inside primers and knockout inside primers.Primers were first validated in both parental cells and bulk selected cells. Results from 24 individual candidate clones are shown together with the expected band patters for wild-type, knockout, and monoallelic deletion clones. The black arrows indicate the correct amplification products, stars indicate biallelic knockout clones.The biallelic mutant lines were further validated by western blot. Nonhomologous end joining uses a single Cas9 cut and selection-independent screening, thus requiring little up-front preparation, and screening for knockout clones by dot blots can quickly determine if a knockout generation was successful. PCR-based screening accurately detects correct candidates with large genomic perturbations generated by homology-directed repair.These techniques enable researchers in molecular biology to easily and efficiently identify knockouts, allowing them to explore normal or dysfunctional cellular processes using cell lines. After watching this video, you should have a good understanding of how to isolate and identify clonal knockout cell lines generated by CRISPR/Cas9 mediated genome editing.

Research

JoVE Journal

Genetics

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

隠すビートルにノックダウン飼育し、二本鎖RNA媒介遺伝子、<em&gt; Dermestes maculatus</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

遺伝学

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format

ハイスループットフォーマットでの遺伝子型CRISPR / Cas9媒介ノックアウト変異体に蛍光PCR-キャピラリーゲル電気泳動法を用いました

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

点突然変異修復 CRISPR/Cas9 とひと細胞での SsODN によって触媒の機能として敷地内変異原性を調べるための標準方法論

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

遺伝子破壊ミュータンス連鎖球菌緊張せず遺伝子クローニングの生成

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in ...

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

CRISPR/Cas9 仲介の対象となる統合生体内で相同性を介した終わり参加ベースの戦略を使用して

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

細胞分裂に CRISPR/Cas9 を介した遺伝子ノックアウトのインテグラーゼ欠損レンチウイルスベクターの生産のためのプロトコル

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells ...

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

効率的な生産とモデル システムの動脈分布に CRISPR 識別/Cas9 生成遺伝子のノックアウト

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development ...

Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells

交配の適応は哺乳類細胞プロテオミクスのクロマチン関連タンパク質の捕捉します。

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein ...

Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells

哺乳類細胞におけるメジャー クロマチン アクセシビリティ規制要素のホルムアルデヒドによる分離

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

CRISPR/Cas9 によるマウスおよび人間の造血前駆細胞の高効率遺伝子破壊

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...