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

Este trabalho foi financiado pelo NIH (R01HL131835), um prêmio de investigador clínico de Damon Runyon e o consórcio de câncer Starr.

1. sgRNA projeto usando ferramentas on-line13
<ol><li>
Projeto sgRNAs como alvo o gene de interesse usando ferramentas disponíveis gratuitamente on-line.
	<ol>
<li>Copie e cole a sequência de referência NCBI do gene de interesse para a ferramenta de web designer do Broad Institute sgRNA: (http://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design). Nota: Esta ferramenta identifica sequências sgRNA com sítios de clivagem dentro exões e aqueles que abrangem ...

<ol>
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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ZFN,+TALEN,+CRISPR/Cas-based+methods+for+genome+engineering.">ZFN, TALEN, CRISPR/Cas-based methods for genome engineering.</a> Trends Biotechnol. 31 (7), 397-405 (2013).</li><li>Jinek, 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=A+programmable+dual-RNA-guided+DNA+endonuclease+in+adaptive+bacterial+immunity.">A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.</a> Science. 337 (6096), 816-821 (2012).</li><li>Wiedenheft, B., Samuel, S. H., Doudna, J. 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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. G., 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=Rational+design+of+highly+active+sgRNAs+for+CRISPR-Cas9-mediated+gene+inactivation.">Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation.</a> Nat Biotechnol. 32, 1262-1267 (2014).</li><li>Brinkman, E. 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=Easy+quantitative+assessment+of+genome+editing+by+sequence+trace+decomposition.">Easy quantitative assessment of genome editing by sequence trace decomposition.</a> Nucl Acid Res. 42, e168 (2014).</li><li>Klampfl, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatic+mutations+of+calreticulin+in+myeloproliferative+neoplasms.">Somatic mutations of calreticulin in myeloproliferative neoplasms.</a> N Engl J Med. 369, 2379-2390 (2013).</li><li>Nangalia, J., 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=Somatic+CALR+mutations+in+myeloproliferative+neoplasms+with+nonmutated+JAK2.">Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2.</a> N Engl J Med. 369, 2391-2405 (2013).</li><li>Elf, 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=Mutant+Calreticulin+requires+both+its+mutant+C-terminus+and+the+thrombopoietin+receptor+for+oncogenic+transformation.">Mutant Calreticulin requires both its mutant C-terminus and the thrombopoietin receptor for oncogenic transformation.</a> Cancer Discovery. 6 (4), 368-381 (2016).</li><li>Bauer, D. 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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>

Aqui vamos mostrar o uso de gene de CRISPR/Cas9 edição para estudar a função biológica de CALR mutações em células hematopoiéticas. O sucesso do presente protocolo é altamente dependente de múltiplos fatores. Em primeiro lugar, é importante saber quais os tipos de mutações podem ser responsáveis para o fenótipo desejado. Neste protocolo, a leitura é a transformação de células Ba/F3 para mIL-3 independência e os tipos de mutações são puntuais no exon 9 de CALR. No entanto, se a mu...

CRISPR/Cas9 tecnologia recentemente revolucionou o genoma alvo edição nos organismos e células vivas. Tornou-se uma ferramenta extremamente poderosa para a investigação biomédica e atualmente está sendo utilizado como uma avenida de potencial para a terapia de doenças genéticas1. A base para todas as ferramentas de edição de genoma baseia-se na criação de uma induzida por nuclease DNA encalhada interrupção dupla (DSB) no locus genômico a ser modificado. Os DSBs podem ser reparados pelo não-homóloga final-adesão (NHEJ) ou reparação de homologia-dirigido (HDR)2,3. A vantagem da nuclease Cas9 sobre outro genoma engenharia nucleases, tais como as nucleases de dedo de zinco (ZFNs) e nucleases efetoras como ativador de transcrição (TALENs) é sua dependência de RNA para o direcionamento da nuclease para uma sequência de DNA desejada, em comparação para as interações proteína-ADN encontradas em ZFNs e TALENs2,3.
Após a descoberta da via de nuclease CRISPR/Cas9 como um sistema imune adaptativo em células procarióticas4,5, muito esforço tem ido para adaptar o caminho para o uso em linhas de células de mamíferos e de organismos modelo2, 3. como uma ferramenta para edição de gene, via CRISPR/Cas9 utiliza dois componentes principais: o Streptococcus pyogenes (Sp) derivado Cas9 nuclease e sgRNAs como alvo o gene de interesse,2,3. O sgRNA consiste de 20 nucleotídeos que Cas9 direto para um site específico sobre o genoma através de pares de base de RNA-DNA a complementaridade2,3. O site de destino do sgRNA devera ser adjacente a um site de motivo adjacentes (PAM) protospacer sob a forma de 5' NGG, que é reconhecido pela nuclease SpCas9. Com estas ferramentas, Cas9 pode ser direcionado para qualquer sequência de DNA através da concepção de sgRNAs destino a região de interesse. Além da Sp derivada Cas9, existem variantes adicionais para Cas9 com características diferentes, dependendo da aplicação específica. Por exemplo, existem variantes de Cas9 com maior especificidade para edição no alvo ou capacidade de clivagem de single-strand DNA roubando6,7. Além disso, recentemente foi desenvolvido Cas9 cataliticamente inactiva por regulamento transcriptional8. Cientistas agora têm usado o sistema CRISPR/Cas9 para uma variedade de aplicações, tais como gene batendo e nocaute para estudar as funções biológicas de genes9, da função de perda e ganho-de-função biblioteca telas10 e genética Engenharia de organismos modelo11.
Neste protocolo, aliamos a metodologia CRISPR/Cas9 com o ensaio de transformação celular Ba/F3 para entender a função biológica do CALR mutações. BA/F3 células são uma linha de células hematopoiéticas dependentes murino IL-3 que pode ser processada IL-3 independente sobre expressão de determinados oncogenes, como BCR-ABL12. A fim de compreender se calreticulin mutante pode transformar células Ba/F3 para crescimento independente de citocina, nós alvo exon 9 do locus endógeno de Calr usando CRISPR/Cas9 para introduzir mutações indel e então IL-3 retirou as células para aplicar um pressão de seleção positiva, com o objetivo de recapitulando mutações de ganho-de-função CALR encontradas em pacientes do MPN. O protocolo inclui o projeto, a clonagem e a entrega de sgRNAs, o desenvolvimento de células expressando de estável Cas9 e triagem para CRISPR gene edição no alvo. Este protocolo pode ser aplicado a diferentes genes e várias linhas de célula dependente de citocinas de interesse e é especialmente valioso em modelagem e estudar a função biológica de genes envolvidos no câncer.

<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

Clusterizado regularmente intercaladas curtas palíndromos repetições (CRISPR) é um sistema de imunidade adaptativa em procariontes que tem sido reaproveitado pelos cientistas para gerar nucleases RNA-guiada, tais como CRISPR-associado (Cas) 9 para site-specific do genoma eucariótico edição. Engenharia de genoma por Cas9 é usada eficientemente, facilmente e robustamente modificar genes endógenos em muitas linhas de células de mamíferos biomedically relevantes e organismos. Aqui mostramos um exemplo de como utilizar a metodologia CRISPR/Cas9 para entender a função biológica de mutações genéticas específicas. Usamos modelos calreticulin (CALR) mutações em murino interleucina-3 (3 mIL) dependentes pro-B (Ba/F3) células pela entrega de RNAs de guia única (sgRNAs) visando o locus Calr endógeno na região específica onde inserção e/ou exclusão (indel ) CALR mutações ocorrem em pacientes com Neoplasias mieloproliferativas (MPN), um tipo de câncer de sangue. Os sgRNAs criar quebras da cadeia dupla (DSBs) na região alvo que são reparadas por fim não-homóloga ingressar (NHEJ) para dar puntuais de vários tamanhos. Então, nós empregamos o ensaio de transformação celular Ba/F3 padrão para entender o efeito da expressão de nível fisiológico de Calr mutações na transformação celular hematopoiética. Esta abordagem pode ser aplicada a outros genes para estudar sua função biológica em várias linhas de células de mamíferos.

Usando o método descrito aqui, o objetivo deste experimento é estudar os efeitos funcionais da introduzir mutações indel para o locus Calr endógena na transformação de células hematopoiéticas. O sistema CRISPR/Cas9 é usado como uma ferramenta para criar endógena Calr mutações em células F3/Ba. Dois sgRNAs foram escolhidos para alvo exon 9 de Calr (Figura 1), na região onde inserções e/ou mutações de exclusão (ind...

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

Gene alvo edição usando CRISPR/Cas9 facilitou muito a compreensão das funções biológicas dos genes. Aqui, nós utilizamos a metodologia CRISPR/Cas9 a mutações de calreticulin modelo em células hematopoiéticas dependentes de citocina para estudar sua atividade oncogênica.

Usando CRISPR/Cas9 Gene edição para investigar a atividade oncogênica do mutante Calreticulin em células hematopoiéticas dependentes de Cytokine

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

o<em&gt; Drosophila</em&gt; Imaginal Modelo Disc Tumor: visualização e quantificação da expressão gênica e invasão tumoral Usando Mosaicos Genéticos

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

Pesquisa do câncer

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

Fabricação de língua matriz extracelular e reconstituição da língua Carcinoma de células escamosas 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

Enriquecimento de Fosfopeptida acoplado com espectrometria de massa livre de rótulo quantitativa para investigar o Phosphoproteome no câncer de próstata

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

Investigação de dependências genéticas utilizando ensaios de competição baseados em 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

Usando CRISPR/Cas9 para knock out GM-CSF em células 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

Detecção de fusão gênica oncogênica usando a reação em cadeia de polimerase multiplex ancorada seguida por sequenciamento da próxima geração

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 e Expansão de células T assassinas induzidas por citocina para tratamento de câncer

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

Mapeando as Relações Estrutura-Função de Fatores de Transcrição Oncogênica Desordenados usando análise transcriômica

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 de Fluidos Proximais para Investigar o Microambiente Tumoral do Adenocarcinoma Pancreático

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 ...