Name: using crispr/cas9 gene editing to investigate the oncogenic activity of mutant calreticulin in cytokine dependent hematopoietic cells
Uploaded: 2018-01-05T15:00:00
Description: Watch this Scientific Journal Video about Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells at JoVE.com

この作品は、NIH (R01HL131835)、デイモン ・ ラニアン臨床賞、スターがんコンソーシアムによって支えられました。

1. sgRNA オンライン ツールを使用してデザイン13
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自由に利用できるオンライン ツールを使用して興味の遺伝子をターゲット sgRNAs をデザインします。
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<li>ブロード研究所 sgRNA デザイナー web ツールに興味の遺伝子の NCBI 参照シーケンスのコピーとペースト: (http://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design)。 注: このツールを識別エクソン内?...

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Q., Baltimore, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+an+interleukin+3-dependent+hematopoietic+cell+line+by+the+chronic+myelogenous+leukemia-specific+bcr/abl+protein.">Transformation of an interleukin 3-dependent hematopoietic cell line by the chronic myelogenous leukemia-specific bcr/abl protein.</a> Proc Natl Acad Sci USA. 85, 9312-9316 (1988).</li><li>Ran, F. 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E., Canver, M. C., Orkin, S. H. <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+Genomic+Deletions+in+Mammalian+Cell+lines+via+CRISPR/Cas9.">Generation of Genomic Deletions in Mammalian Cell lines via CRISPR/Cas9.</a> J. Vis. Exp. (95), e52118 (2015).</li><li>Watanabe-Smith, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+acquired+mutations+in+transgenes+arising+in+Ba/F3+transformation+assays:+findings+and+recommendations.">Analysis of acquired mutations in transgenes arising in Ba/F3 transformation assays: findings and recommendations.</a> Oncotarget. 8, 12596-12606 (2017).</li><li>Fu, Y., 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=Improving+CRISPR-Cas+nuclease+specificity+using+truncated+guide+RNAs.">Improving CRISPR-Cas nuclease specificity using truncated guide RNAs.</a> NatBiotechnol. 32, 279-284 (2014).</li></ol>

ここで我々 は造血細胞のCALR突然変異の生物学的機能を研究する CRISPR/Cas9 遺伝子編集の使用を示します。このプロトコルの成功は複数の要因に依存します。まず、どのような種類の突然変異は、望まれる表現型の責任を知ることが重要です。このプロトコルでは読み出しはミル 3 独立 ba/f3 細胞のトランスフォーメーションと突然変異の種類です。 CALRのエクソン 9 オクターリピ?...

CRISPR/Cas9 技術最近生きている細胞および生物における標的ゲノム編集をもたらしました。それは生物医学研究のための非常に強力なツールとなっている、現在遺伝病1の治療のための潜在的な道として利用されています。すべてのゲノムの編集ツールのための基礎は変更する genomic 位置で、ヌクレアーゼによる DNA 二重鎖切断 (DSB) の作成に依存します。非相同末端結合 (NHEJ) または相同監督修理 (HDR)2,3によって、Dsb を修復できます。他のゲノムの核酸、亜鉛指核酸 (ZFNs) などを工学上 Cas9 ヌクレアーゼと転写活性化因子のようなエフェクター核酸分解酵素 (TALENs) の利点は、比較目的の DNA シーケンスにヌクレアーゼのターゲット RNA 依存性ZFNs と TALENs2,3の蛋白質 DNA 相互作用。
原核細胞4,5適応免疫系として CRISPR/Cas9 ヌクレアーゼ経路の発見は後、多くの努力は哺乳類セルライン、モデル生物2,用経路の適応になっています。3。 遺伝子編集用ツールとして CRISPR/Cas9 経路は 2 つの主要なコンポーネントを利用して: Cas9 ヌクレアーゼと sgRNAs 金利2,3の遺伝子をターゲットに化膿連鎖球菌(Sp) が派生しました。SgRNA は、20 ヌクレオチド RNA DNA 塩基の相補性2,3ゲノムの特定のサイトに、直接 Cas9 で構成されます。SgRNA のターゲット サイトは 5' SpCas9 ヌクレアーゼによって認識され、NGG の形で protospacer の隣接するモチーフ (PAM) のサイトに隣接してある必要があります。これらのツールは、sgRNAs を対象とする関心領域を設計することによって任意の DNA シーケンスに Cas9 を送信できます。さらに Sp Cas9 を得、特定のアプリケーションによって異なる機能を持つ Cas9 の追加バリエーションがあります。たとえば、対象の編集に高い特異性と6、7の刻み目をつける人 DNA の単鎖切断能力 Cas9 バリエーションがあります。さらに、触媒非アクティブ Cas9 は最近転写調節8に開発され。科学者は、遺伝子のノックやノックアウト遺伝子9、機能喪失、利得関数のライブラリ画面10と遺伝子の生物学的機能を研究するなど、アプリケーションのさまざまな CRISPR/Cas9 システムを使用している今モデル生物11工学。
このプロトコルではCALRの突然変異の生物学的機能を理解する、ba/f3 細胞形質転換試験 CRISPR/Cas9 の方法論と組み合わせています。Ba/f3 細胞は IL 3 に表示できるマウス IL 3 依存造血細胞ライン BCR-ABL12など特定の遺伝子の発現に依存します。変異カルレティキュリン ba/f3 細胞サイトカイン依存性の成長を変換できるかどうかを理解するためには、CRISPR/Cas9 を使用して塩基変異を導入する内因性のCalr座のエクソン 9 を対象とし、適用するセルから IL 3 を撤回した、正の選択圧、MPN 患者は、利得関数のCALR突然変異をさたことを目標にします。デザイン、クローニング、sgRNAs、Cas9 の安定発現細胞の開発の配信プロトコルが含まれ、CRISPR 検診対象の遺伝子編集します。このプロトコルは異なる遺伝子または様々 なサイトカイン依存性細胞線に適用できるし、モデリングと癌関連遺伝子の生物学的機能を勉強で特に有用。

<table>
	<tbody>
		<tr>
			<td>BsmBI</td>
			<td>New England Biolabs</td>
			<td>R0580L</td>
			<td></td>
		</tr>
		<tr>
			<td>Blasticidin</td>
			<td>Sigma Aldrich</td>
			<td>15025</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin</td>
			<td>Life Technologies</td>
			<td>A1113803</td>
			<td></td>
		</tr>
		<tr>
			<td>Stbl3 cells</td>
			<td>Life Technologies</td>
			<td>C737303</td>
			<td></td>
		</tr>
		<tr>
			<td>TransIT-LT1</td>
			<td>Thermo Fisher Scientific</td>
			<td>MIR2300</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Life Technologies</td>
			<td>51985034</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Thermo Fisher Scientific</td>
			<td>MT10040CV</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>10-017-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Omega Scientific</td>
			<td>FB-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin/L-glutamine</td>
			<td>Life Technologies</td>
			<td>10378016</td>
			<td></td>
		</tr>
		<tr>
			<td>mIL-3</td>
			<td>Peprotech</td>
			<td>213-13</td>
			<td></td>
		</tr>
		<tr>
			<td>psPAX2</td>
			<td>Addgene</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>pCMV-VSV-G</td>
			<td>Addgene</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>pLX_TRC311-Cas9</td>
			<td>Addgene</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>polybrene</td>
			<td>Sigma Aldrich</td>
			<td>H9268</td>
			<td></td>
		</tr>
		<tr>
			<td>pXPR-011</td>
			<td>Addgene</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Genessee Scientific</td>
			<td>25-507</td>
			<td></td>
		</tr>
		<tr>
			<td>TAE buffer</td>
			<td>Thermo Fisher Scientific</td>
			<td>FERB49</td>
			<td></td>
		</tr>
		<tr>
			<td>LentiGuide-Puro</td>
			<td>Addgene</td>
			<td>Plasmid #52963</td>
			<td></td>
		</tr>
		<tr>
			<td>PNK</td>
			<td>New England Biolabs</td>
			<td>M0201S</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 ligase</td>
			<td>New England Biolabs</td>
			<td>M0202S</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR purification kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td></td>
		</tr>
		<tr>
			<td>Miniprep kit</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td></td>
		</tr>
	</tbody>
</table>

using crispr/cas9 gene editing to investigate the oncogenic activity of mutant calreticulin in cytokine dependent hematopoietic cells

クラスター化された空間定期的に短い回文繰り返し (CRISPR) は CRISPR 関連 (Cas) 9 サイト固有の真核生物ゲノムのなどの RNA 誘導の核酸を生成する科学者によって用途が変更されている原核生物の適応免疫システム編集します。Cas9 によるゲノム工学を使用して、効率的に、簡単に、確実が多く biomedically に関連する哺乳類細胞および生物における内在性の遺伝子を変更します。ここで特定の遺伝の突然変異の生物学的機能を理解する CRISPR/Cas9 方法論を利用する方法の例を紹介します。シングル ガイド Rna (sgRNAs) 特定の地域における内因性Calr座をターゲットの配信によってカルレティキュリン (CALR) 変異によるインターロイキン 3 (mIL-3) 依存プロ B (Ba/F3) 細胞内モデル、挿入・削除 (塩基) 骨髄増殖性腫瘍 (MPN)、血液の癌の種類と患者のCALRの突然変異が発生します。SgRNAs は、非相同末端結合 (NHEJ) 様々 なサイズの indels の扱いを与えることで修復されている対象地域の二重鎖切断 (Dsb) を作成します。我々 は、造血細胞変換のCalr突然変異の生理的レベル式の効果を理解する、標準 ba/f3 細胞形質転換試験を採用しています。このアプローチは、様々 な哺乳類細胞の生物学的機能を研究する他の遺伝子に適用できます。

ここで説明した方法を使用して、この実験の目的は、内因性造血細胞形質転換Calr遺伝子座に塩基変異導入機能の効果です。CRISPR/Cas9 システムは、ba/f3 細胞の内因性のCalr突然変異を作成するツールとして使用されます。2 sgRNAs は、 CALRで通常発生の挿入または削除 (塩基) 変異領域でエクソン 9 のCalr (図 1) をターゲット?...

Watch this Scientific Journal Video about Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells at JoVE.com

Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells

CRISPR/Cas9 を使用して、編集対象となる遺伝子は、遺伝子の生物機能の理解を促進しています。ここでは、その発癌性の活動を研究するためにサイトカイン依存性造血細胞のモデル カルレティキュリン変異に CRISPR/Cas9 方法論を活用します。

CRISPR/Cas9 を使用して遺伝子変異カルレティキュリン サイトカイン依存性造血細胞での発癌性の活動を調査する編集

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to ...

The overall goal of this experiment is to utilize CRISPR/Cas9 gene editing to model calreticulin mutations in cytokine dependent hematopoietic cells in order to study their oncogenic activity. This method can help answer key questions in the hematological malignancies field such as the biological function of driver mutations when they are expressed at physiological levels. The main advantage of this technique is that it utilizes the CRISPR/Cas9 gene editing system to model and study the biological function of genes involved in cancer.To anneal and phosphorylate the oligos, add the reagents to a total of 10 microliters in a PCR tube kept on ice. Set the program in the thermocycler. Use water to dilute the reaction after cycle is complete.To digest the lentiGuide-Puro vector, prepare 50 microliters of digestor reaction. Then, start the digestion reaction at 55 degrees Celsius at a heat block. After one hour, add an additional microliter of BsmB1 restriction enzyme and continue the reaction for another hour.To dephosphorylate the cut back bone, add seven microliters of phosphatase reaction buffer and two microliters of phosphatase enzyme to the digestive vector. Then, incubate the mixture at 37 degrees Celsius for 30 minutes. Purify the already cut and dephosphylated back bone using a commercial kit.To prepare the ligation mixture, add 15 nanograms of digested back bone and other reagents to a PCR tube kept on ice and adjust the volume to 10 microliters with water. Incubate the reaction at room temperature for one hour. Then transform STBL3 compatible cells with two microliters of the ligation mixture.And then plate on the lysogeny broth agar plate containing 100 micrograms per milliliter of ampicillin. After this, incubate the plate overnight at 37 degrees Celsius. Pick three colonies and inoculate them.Perform mini prep for each culture and sequence the oligo insertion site with a U6 promoter region primer. Seed hek293t cells on a 10 centimeter dish. And leave it for overnight incubation.To prepare the DNA mixture for transfection, pipette 45 microliters of the transfection reagent to the DNA. Use a pipette to mix the DNA and transfection reagent carefully, and allow it to stand for 20 minutes. After this, add the DNA transfection mixture drop-wise on the seeded cells.Gently swirl the plates. And incubate at 5%carbon dioxide and 37 degrees Celsius for a day. Harvest the virus containing supernatent after 24 and 48 hours by passing it through a 0.22 micromiter filter.And aliquot 1.6 milliliters of supernatent in each cryo vial. Next aspirate the supernatent and resuspend the Ba/F3 cells in media by pipetting the cells up and down several times. Add 500 microliters of the resuspended cells, 1.5 milliliters of viral supernatant, four microliters of polybrene, and 10 nanograms per milliliter of murine interleukin 3 in a six well tissue culture plate.Centrifuge the plate at 440 G for 120 minutes at 37 degrees Celsius. After centrifugation, transfer the plate to an incubator for overnight incubation at 37 degrees Celsius with 5%carbon dioxide. After this, spin the cells down at 440 G for three minutes.And resuspend in five milliliters of fresh warm media in a six well plate. To select the Cas9 infected cells, first spin down the cell. And then resuspend in fresh warm media supplemented with five micrograms per milliliter of blasticidin after 48 hours of spinfection.Continue selecting cells for nine days with blasticidin til all the uninfected cells are dead. Then, transfer the resistant cells to a medium with one microgram per milliliter of blasticidin. Transduce the parental cells and the cells stably expressing Cas9 with pXPR-011 reporter construct through lentiviral infection.Continue selecting cells with two micrograms per milliliter of puromycin for three days, 48 hours after spinfection. Infect the Cas9 expressing cells with single guide ribonucleic acid using lentivirus. Then, use two micrograms per milliliter of puromycin to select the cells for three days, 48 hours post spinfection.Bin the exponentially growing Ba/F3 cells expressing ectopic Cas9 and the single guide ribonucleic acid of interest. Aspirate the supernatent and wash the cells with five milliliters of phosphate buffered saline. Spin the cells down, and wash with phosphate buffered saline three times.Then, count the number of cells using a cell viability analyzer. After this, seed cells in triplicates in two milliliters of fresh media without interleukin 3 in six well culture plates. Start monitoring and counting the cells every two days for eight days.Representative flow psychometric data demonstrating Cas9 activity in parental Ba/F3 cell and Ba/F3 overexpressing Cas9 cells is shown here. Green fluorescent protein signal is reduced by 76%in Ba/F3 overexpressing Cas9 cells, indicating robust Cas9 activity, whereas in parental Ba/F3 cells, the green fluorescent protein signal remains at 100%Representative growth curves for cells demonstrating oncogenic activity of calreticulin are shown here. CRISPR/Cas9 mediated plus one base pair frame shift mutation was introduced in the exon nine locus of calreticulin using m1 and m2 single guide RNase.Growth curves for calreticulin targeted Ba/F3 thrombopoietin receptor Cas9 cells shows murine interleukin 3 independent growth. However, the growth curves for the other cell types do not show any cytokine independent growth. Sequencing results confirm the on target editing of endogynous calreticulin exon nine in calreticulin targeted Ba/F3 thrombopoietin receptor Cas9 cells using the m1 and m2 single guide RNase.The plus one base pair frame shift mutations are indicated in black. Once mastered, this technique can be done in two weeks if performed properly. While attempting this procedure, it's important to understand what kind of mutations in the gene of interest may be responsible for the phenotype that is desired.Following this procedure, other methods to further characterize the mutation, such as its role in cell signaling or in protein protein interactions can be performed in order to further understand the mechanism by which is causes disease. After its development, this technique paved the way for researchers in the field of hematology to explore the effect of gain-of-function mutations at physiological levels on cell transformation in Ba/F3 hematopoietic cells. After watching this video, you should have a good understanding of how to utilize the CRISPR/Cas9 gene editing methodology to investigate the oncogenic activity of gain-of-function mutations in cytokine dependent hematopoietic cells.Don't forget that working with lentivirus can be extremely hazardous and precautions such as working in a BL2 plus facility should always be taken while performing this procedure.

Research

JoVE Journal

Cancer Research

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

ザ<em&gt;ショウジョウバエ</em&gt;成虫盤腫瘍モデル：遺伝的モザイクを用いた遺伝子発現と腫瘍侵襲性の可視化と定量化

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

がん研究

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

舌の細胞外マトリックスと舌扁平上皮癌生体外の再構成の作製

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce ...

Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer

前立腺癌におけるリン酸化プロテオームを調べる定量的ラベル無料質量と相まってリン酸化ペプチド濃縮

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction ...

Investigation of Genetic Dependencies Using CRISPR-Cas9-based Competition Assays

CRISPR ベース Cas9 の競争の試金を使用して遺伝的依存関係の調査

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene ...

Using CRISPR/Cas9 to Knock Out GM-CSF in CAR-T Cells

CRISPR/Cas9を使用してCAR-TセルのGM-CSFをノックアウト

Chimeric antigen receptor T (CAR-T) cell therapy is a cutting edge and potentially revolutionary new treatment option for cancer. However, there are ...

Oncogenic Gene Fusion Detection Using Anchored Multiplex Polymerase Chain Reaction Followed by Next Generation Sequencing

アンカー多重ポリメラーゼ連鎖反応を用いた腫瘍性遺伝子融合検出、次世代シーケンシング

Gene fusions frequently contribute to the oncogenic phenotype of many different types of cancer. Additionally, the presence of certain fusions in samples ...

Isolation and Expansion of Cytotoxic Cytokine-induced Killer T Cells for Cancer Treatment

癌治療のための細胞傷害性サイトカイン誘発キラーT細胞の分離と拡張

Adoptive cellular immunotherapy focuses on restoring cancer recognition via the immune system and improves effective tumor cell killing. Cytokine-induced ...

Mapping the Structure-Function Relationships of Disordered Oncogenic Transcription Factors Using Transcriptomic Analysis

トランスクリプトーム解析を用いた無秩序発がん性転写因子の構造機能関係のマッピング

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these ...

Isolation of Proximal Fluids to Investigate the Tumor Microenvironment of Pancreatic Adenocarcinoma

膵臓腺癌の腫瘍微小環境を調べるための近位液の分離

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables ...

CRISPR/Cas9 を使用して遺伝子変異カルレティキュリン サイトカイン依存性造血細胞での発癌性の活動を調査する編集

CRISPR/Cas9 を使用して遺伝子変異カルレティキュリンサイトカイン依存性造血細胞での発癌性の活動を調査する編集