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

Questo lavoro è stato supportato da NIH (R01HL131835), un premio di ricercatore clinico di Damon Runyon e il Consorzio di cancro Starr.

1. sgRNA Design utilizzando strumenti Online13
<ol><li>
Progettazione sgRNAs targeting per il gene di interesse utilizzando gli strumenti disponibili gratuitamente online.
	<ol>
<li>Copiare e incollare la sequenza di Riferimento NCBI del gene di interesse lo strumento di progettazione web sgRNA Broad Institute: (http://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design). Nota: Questo strumento identifica sgRNA sequenze con siti di taglio all'interno degli esoni...

<ol>
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S., 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=CRISPR/Cas9-mediated+Gene-knockout+screens+and+target+identification+via+Whole+genome+sequencing+uncover+host+genes+required+for+picornavirus+infection.">CRISPR/Cas9-mediated Gene-knockout screens and target identification via Whole genome sequencing uncover host genes required for picornavirus infection.</a> J Biol Chem. 10, 1074 (2017).</li><li>Heckl, D., 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=Generation+of+mouse+models+of+myeloid+malignancies+with+combinatorial+genetic+lesions+using+CRISPR-Cas9+genome+editing.">Generation of mouse models of myeloid malignancies with combinatorial genetic lesions using CRISPR-Cas9 genome editing.</a> Nat Biotechnol. 32 (9), 941-946 (2014).</li><li>Daley, G. 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. A., 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=Genome+engineering+using+the+CRISPR-Cas9+system.">Genome engineering using the CRISPR-Cas9 system.</a> Nat Protoc. 8 (11), 2281-2308 (2013).</li><li>Haeussler, 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=Evaluation+of+off-target+and+on-target+scoring+algorithms+and+integration+into+the+guide+RNA+selection+tool+CRISPOR.">Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR.</a> Genome Biol. 17, 148 (2016).</li><li>Doench, J. 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(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>

Qui dimostriamo l'uso di CRISPR/Cas9 gene editing per studiare la funzione biologica di CALR mutazioni nelle cellule ematopoietiche. Il successo di questo protocollo è altamente dipendente da molteplici fattori. In primo luogo, è importante sapere quali tipi di mutazioni possono essere responsabili del fenotipo che è desiderato. In questo protocollo, la lettura è la trasformazione delle cellule Ba/F3 per mIL-3 indipendenza e i tipi di mutazioni sono indels in essone 9 di CALR. Tuttavia, se la mutazi...

CRISPR/Cas9 tecnologia recentemente ha rivoluzionato l'editing genomico mirato negli organismi e cellule viventi. È divenuto uno strumento estremamente potente per la ricerca biomedica e attualmente viene utilizzato come un viale potenziale per la terapia di malattie genetiche1. La base per tutti gli strumenti di editing del genoma si basa sulla creazione di una nucleasi-indotta rottura del DNA doppia filamento (DSB) presso il locus genomico per essere modificato. Il DSBs può essere riparato da fine-unirsi non-omologo (NHEJ) o riparazione omologia-diretto (HDR)2,3. Il vantaggio della nucleasi Cas9 sopra altri genoma ingegneria nucleasi, quali nucleasi a dita di zinco (ZFNs) e nucleasi di trascrizione attivatore-come effettore (TALENs) è la dipendenza dal RNA per il targeting della nucleasi a una sequenza di DNA desiderata, rispetto le interazioni proteina-DNA trovato in ZFNs e TALENs2,3.
Dopo la scoperta del pathway di nucleasi CRISPR/Cas9 come un sistema immunitario adattativo in cellule procariotiche4,5, molto sforzo è andato in adattando la via per l'uso in linee cellulari di mammifero e modello organismi2, 3. come strumento per l'editing di gene, il pathway CRISPR/Cas9 utilizza due componenti principali: Streptococcus pyogenes (Sp) derivato Cas9 nucleasi e sgRNAs del gene di interesse2,3di targeting. Il sgRNA è costituito da 20 nucleotidi che Cas9 diretto a un sito specifico sul genoma a RNA-DNA base pair complementarità2,3. Il sito di destinazione del sgRNA deve trovarsi adiacente a un sito di motivi adiacenti (PAM) protospacer sotto forma di 5' NGG, che è riconosciuto dalla nucleasi SpCas9. Con questi strumenti, Cas9 può essere diretto a qualsiasi sequenza di DNA con la progettazione di sgRNAs che hanno come destinazione la regione di interesse. Inoltre, Sp derivato Cas9, esistono varianti supplementari per Cas9 con caratteristiche diverse a seconda dell'applicazione specifica. Ad esempio, ci sono varianti di Cas9 con maggiore specificità per l'editing on target o capacità di single-strand fenditura per DNA intaccare6,7. Inoltre, cataliticamente inattivo Cas9 recentemente è stata sviluppata per regolazione trascrizionale8. Gli scienziati ora hanno utilizzato il sistema CRISPR/Cas9 per una varietà di applicazioni, ad esempio Knockin ' genica e knockout per studiare le funzioni biologiche di geni9, perdita di funzione e guadagno di funzione libreria schermi10 e genetica Ingegneria di organismi modello11.
In questo protocollo, uniamo la metodologia CRISPR/Cas9 con il saggio di trasformazione cellulare Ba/F3 a capire la funzione biologica di CALR mutazioni. Le cellule Ba/F3 sono una linea di cellule ematopoietiche dipendente dal-3 murina che può essere reso IL-3 indipendente sull'espressione di alcuni oncogeni come BCR-ABL12. Al fine di comprendere se calreticulina mutante può trasformare le cellule Ba/F3 alla citochina crescita indipendente, abbiamo mirato dell'essone 9 del locus endogeno Calr usando CRISPR/Cas9 per introdurre le mutazioni indel e quindi si ritirò IL-3 dalle cellule per applicare un pressione di selezione positiva, con l'obiettivo di guadagno di funzione CALR le mutazioni trovate in pazienti MPN di ricapitolare. Il protocollo comprende la progettazione, la clonazione e la consegna di sgRNAs, lo sviluppo di cellule esprimenti Cas9 stabili e screening per CRISPR gene editing on target. Questo protocollo può essere applicato a diversi geni e varie linee di citochina-dipendente delle cellule di interesse ed è particolarmente prezioso nella modellazione e studiando la funzione biologica di geni coinvolti nel cancro.

<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

Cluster regolarmente intercapedine ripetizioni brevi palindromi (CRISPR) è un sistema di immunità adattativa nei procarioti che è stato reimpiegato dagli scienziati per generare nucleasi RNA-guidato, come ad esempio CRISPR-collegata (Cas) 9 per site-specific genoma eucariotico di editing. Ingegneria del genoma da Cas9 viene utilizzato per in modo efficiente, robusta e facilmente modificare geni endogeni in molte linee cellulari di mammifero biomediche pertinenti e organismi. Qui vi mostriamo un esempio di come utilizzare la metodologia CRISPR/Cas9 per comprendere la funzione biologica di mutazioni genetiche specifiche. Abbiamo il modello calreticulina (CALR) mutazioni in murino interleukin-3 (mIL-3) pro-B (Ba/F3) le celle dipendenti dalla consegna di guida singola RNAs (sgRNAs) targeting per il locus Calr endogeno nella regione specifica dove inserimento e/o cancellazione (indel ) CALR mutazioni si verificano in pazienti con neoplasie mieloproliferative (MPN), un tipo di tumore del sangue. La sgRNAs creare doppio filo (DSBs) nella regione mirata che sono riparati da non-omologo fine unirsi (NHEJ) per dare indels di varie dimensioni. Utilizziamo quindi il test di trasformazione cellulare Ba/F3 standard per comprendere l'effetto dell'espressione di livello fisiologico di Calr mutazioni sulla trasformazione cellulare ematopoietico. Questo approccio può essere applicato ad altri geni di studiare la loro funzione biologica in varie linee cellulari di mammifero.

Utilizzando il metodo descritto qui, l'obiettivo di questo esperimento è quello di studiare gli effetti funzionali dell'introduzione di mutazioni indel al locus Calr endogeno sulla trasformazione delle cellule ematopoietiche. Il sistema CRISPR/Cas9 è usato come uno strumento per creare endogeno Calr mutazioni nelle cellule Ba/F3. Due sgRNAs sono stati scelti per indirizzare essone 9 di Calr (Figura 1), nella regione dove gli inser...

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

Genica mirata di editing con CRISPR/Cas9 ha notevolmente facilitato la comprensione delle funzioni biologiche dei geni. Qui, utilizziamo la metodologia CRISPR/Cas9 a modello calreticulina mutazioni in cellule ematopoietiche citochina-dipendente al fine di studiare la loro attività oncogena.

Utilizzando CRISPR/Cas9 Gene Editing per studiare l'attività oncogena di calreticulina mutante in cellule ematopoietiche Cytokine dipendente

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

Il<em&gt; Drosophila</em&gt; Immaginale Disc tumore Modello: visualizzazione e la quantificazione di espressione genica e di Tumor invasività Utilizzando genetica Mosaici

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

Ricerca

Ricerca sul cancro

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

Fabbricazione di lingua della matrice extracellulare e la ricostituzione di Carcinoma di cellule Squamous Tongue In Vitro

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

Fosfopeptide arricchimento accoppiata alla spettrometria di massa privo di etichetta quantitativa per indagare il Phosphoproteome nel carcinoma della prostata

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

Indagine delle dipendenze genetiche utilizzando dosaggi di concorrenza basati su CRISPR-Cas9

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

Utilizzo di CRISPR/Cas9 per eliminare GM-CSF nelle celle CAR-T

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

Rilevamento oncogenico della fusione genica utilizzando la reazione della catena di polimerasi multiplex ancorata seguita dal sequenziamento di nuova generazione

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

Isolamento ed espansione delle cellule T Killer citotokine indotte da citokine per il trattamento del cancro

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

Mappatura delle relazioni struttura-funzione dei fattori di trascrizione oncogenica disordinati utilizzando l'analisi trascrittomica

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

Isolamento di fluidi prossimali per studiare il microambiente tumorale dell'adenocarcinoma pancreatico

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 ...