Name: Knockout genico mediato da CRISPR-Cas9 basato sullelettroporazione in cellule THP-1 e isolamento di cloni a singola cellula (Video) | JoVE Journal
Uploaded: 2025-02-28T12:30:00.000+00:00
Duration: 9 min 29 s
Description: Impara a modificare geneticamente le cellule THP-1 con CRISPR-Cas9 per studiare la resistenza dei macrofagi all'infezione da HIV. Questo protocollo include l'elettroporazione e l'isolamento di cloni a singola cellula.

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</ol>

<table><tbody><tr><td>0,2 &micro; m filtro per siringa</td><td>ClearLine</td><td>146560</td><td>_</td></tr><tr><td>0,4 % blu di tripano</td><td>Beckman Coulter</td><td>383200</td><td>_</td></tr><tr><td>1,5 mL provetta</td><td>Eppendorf</td><td>3810X</td><td>_</td></tr><tr><td>piastra a 24 pozzetti</td><td>Corning</td><td>353047</td><td>_</td></tr><tr><td>6x TriTrack DNA Loading Dye</td><td>Thermo scientific</td><td>R1161</td><td>_</td></tr><tr><td>75 cm&sup2; Pallone di coltura Tappo ventilato</td><td>Corning</td><td>353136</td><td>_</td></tr><tr><td>8 strisce Provette per PCR con tappi Life</td><td>technologies</td><td>AM12230</td><td>_Piastre</td></tr> a <tr><td>96 pozzetti Corning a fondo piatto</td> Piastre <td>a</td><td>353072</td><td>_</td></tr><tr><td>96 pozzetti Corning a fondo tondo</td><td>Corning</td><td>353077</td><td>_</td></tr><tr><td>Agarose</td><td>Euromedex</td><td>D5</td><td>_</td></tr><tr><td>ATGpr</td><td>_</td><td>_</td><td>https://atgpr.dbcls.jp/</td></tr> <tr><td>Sistema di imaging ChemiDoc</td><td>BIO-RAD</td><td></td> <td>12003153_</td></tr><tr><td>Vetrino di conteggio</td><td>NanoEntek</td><td>DHC-N04</td><td>_</td></tr><tr><td>CRISPOR</td><td>_</td><td>_</td><td>http://crispor.gi.ucsc.edu/</td></tr> <tr><td>DPBS</td><td>Gibco</td><td>14190094</td><td>_</td></tr><tr><td>Ensembl</td><td>EMBL-EBI_</td><td></td><td>https://www.ensembl.org/index.html</td></tr><tr><td>Fetale Bovino Siero</td><td>Sigma-Aldrich</td><td>F7524</td><td>_</td></tr><tr><td>FlowJo</td><td>BD Life Sciences</td><td>v10.10</td><td>_</td></tr><tr><td>GeneRuler 100 bp Plus DNA Ladder</td><td>Thermo scientific</td><td>SM0323</td><td>_</td></tr><tr><td>Genome Data Viewer</td><td>NCBI</td><td>_</td><td>https://www.ncbi.nlm.nih.gov/gdv/</td></tr> <tr><td>GraphPad Prism</td><td>Dotmatics</td><td>_</td><td>Versione 9.3.1</td></tr><tr><td>Herculase II Fusion DNA Polimerasi</td><td>Agilent</td><td>600679</td><td>_</td></tr><tr><td>ICE Strumento di analisi CRISPR</td><td>Synthego</td><td>_</td><td>https://www.synthego.com/products/bioinformatics/crispr-analysis</td></tr><tr><td>Image Lab Touch</td><td>BIO-RAD</td><td>_</td><td>Versione 2.4.0.03</td></tr><tr><td>NEBuffer 2</td><td>New England Biolabs</td><td>B7002S</td><td>Incluso con T7EI M0302S</td></tr><tr><td>Neon Kit, 10 &; L</td> Kit di elettroporazione <td>Invitrogen</td><td>MPK1025K</td><td>contenente punte, provette, tampone R ed E</td></tr><tr><td>Sistema di trasfezione al neon</td><td>Invitrogen</td><td>MPK5000</td><td>_</td></tr><tr><td>NetStart 1.0</td><td>_</td><td>_</td><td>https://services.healthtech.dtu.dk/services/NetStart-1.0/</td></tr> <tr><td>Sintetizzatore d'acqua senza nucleasi</td><td></td><td>_</td><td>_</td></tr><tr><td>Primer PCR (EGFP)</td><td>Eurofins</td><td>_</td><td>Fw : GGAATGCAAGGTCTGTTGAATG ; Rev : CACCTTGATGCCGTTCTTCT</td></tr><tr><td>PCR primer (SAMHD1)</td><td>Eurofins</td><td>_</td><td>Fw : CGGGATTGATTTGAGGACGA ; Rev : GGGTGGCAAGTTAGTGAAGA</td></tr><tr><td>Penicillina-streptomicina (10.000 U/mL)</td><td>Gibco</td><td>15140122</td><td>_</td></tr><tr><td>PFA</td><td>Scienze della microscopia elettronica</td><td>15714</td><td>_</td></tr><tr><td>PMA</td><td>Sigma-Aldrich</td><td>P8139</td><td>_</td></tr><tr><td>PrimerQuest</td><td>IDT</td><td>_</td><td>https://eu.idtdna.com/pages/tools/primerquest</td></tr><tr><td>Kit di purificazione QIAquick PCR</td><td>Qiagen</td><td>28104</td><td>_</td></tr><tr><td>Soluzione di estrazione del DNA QuickExtract</td><td>Biosearch Technologies</td><td>QE09050</td><td>_</td></tr><tr><td>RPMI 1640, GlutaMAX</td><td>Gibco</td><td>61870010</td><td>_</td></tr><tr><td>SnapGene Viewer</td><td>Dotmatics</td><td>_</td><td>Version 7</td></tr><tr><td>SpCas9 2NLS Nuclease</td><td>Synthego</td><td>_</td><td>_</td></tr><tr><td>SYBR Safe DNA Gel Stain</td><td>Invitrogen</td><td>S33102</td><td>_</td></tr><tr><td>Synthetic sgRNA (EGFP)</td><td>Synthego</td><td>_</td><td>#1 : CGCGCCGAGGUGAAGUUCGA ; #2 : UUCAAGUCCGCCAUGCCCGA ; #3 : CAACUACAAGACCCGCGCCG</td></tr><tr><td>Sintetico sgRNA (SAMHD1)</td><td>Synthego</td><td>_</td><td>#1 : AUCGCAACGGGGGGCUUGG ; #2 : GCAGUCAAGAACCUCGGCGC ; #3 : CCAUCCCGACUACAAGACAU</td></tr><tr><td>Siringa Plastipak Luer Lock</td><td>BD</td><td>301229</td><td>_</td></tr><tr><td>T100 Termociclatore</td><td>BIO-RAD</td><td>1861096</td><td>_</td></tr><tr><td>T7 endonucleasi I</td><td>Nuovo Inghilterra Biolabs</td><td>M0302S</td><td>_</td></tr><tr><td>TAE tampone UltraPure, 10x</td><td>Invitrogen</td><td>15558026</td><td>400 mM Tris-Acetato, 10 mM EDTA</td></tr><tr><td>THP-1 cellule</td><td>ATCC</td><td>TIB-202</td><td>_</td></tr><tr><td>Tripsina-EDTA (0,05 %)</td><td>Gibco</td><td></td> <td>25300054_</td></tr><tr><td>ZE5 Cell Analyzer</td><td>BIO-RAD</td><td>12014135</td><td></td></tr></tbody></table>_

knockout genico mediato da crispr-cas9 basato sull'elettroporazione in cellule thp-1 e isolamento di cloni a singola cellula

Electroporation-Based CRISPR-Cas9-Mediated Gene Knockout in THP-1 Cells and Single-Cell Clone Isolation

Una linea cellulare THP-1 è stata generata esprimendo stabilmente la proteina reporter GFP (THP-1_GFP) (Figura 2A) e utilizzata come strumento per stabilire un protocollo per un'efficiente edizione genica mediata da CRISPR-Cas9. A questo scopo, 3 sgRNA mirati al gene EGFP sono stati progettati con lo strumento web CRISPOR29 (Figura 2B), che sono stati simultaneamente complessati con Cas9 a un rapporto molare di 9:1 per formare RN...

Knockout genico mediato da CRISPR-Cas9 basato sull'elettroporazione in cellule THP-1 e isolamento di cloni a singola cellula

The human acute monocytic leukemia (AML) THP-1 cell line is widely used as a model to study the functions of human monocyte-derived macrophages, including ...

electroporation-based-crispr-cas9-mediated-gene-knockout-thp-1-cells

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JoVE Journal

Immunology and Infection

Immunologia e Infezioni

In questo video, dimostriamo l'editing genomico all-in-one basato su CRISPR-Cas9 in cellule in coltura in cui Cas9 e sgRNA sono forniti come un singolo costrutto plasmidico alle cellule. Il sistema CRISPR-Cas9 e il gene desiderato da inserire sono stati introdotti nelle cellule attraverso la tecnica dell'elettroporazione per facilitare il successo dell'editing genetico.

Editing genomico CRISPR all-in-one: un metodo per il knock-in genico basato sulla riparazione diretta dall'omologia in cellule coltivate utilizzando il sistema CRISPR-Cas9

Questo video mostra un test per l'esecuzione dell'editing genetico in cellule T umane utilizzando la tecnologia CRISPR-Cas9. Una miscela di cellule T primarie CD4+ e CD8+ è combinata con un complesso ribonucleoproteico CRISPR-Cas9, mirato a geni specifici per il knockout. Dopo l'elettroporazione, l'sgRNA guida Cas9 verso la sequenza di DNA bersaglio, creando tagli precisi. Questi tagli vengono poi riparati dal meccanismo di giunzione non omologa della cellula, portando al knockout genico.

Una tecnica CRISPR-Cas9 per l'editing genetico nelle cellule T

La tecnica qui descritta genotipizzazione, che accoppia fluorescente reazione a catena della polimerasi (PCR) per elettroforesi su gel capillare, permette high-throughput genotipizzazione di cloni knockout nucleasi-mediata. Elude limitazioni incontrate da altre tecniche di genotipizzazione ed è più conveniente rispetto ai metodi di sequenziamento.

Usando una tecnica fluorescente PCR-capillare elettroforesi su gel al genotipo CRISPR / Cas9 mediata Knockout Mutants in un formato high-throughput

I recenti progressi nella capacità di manipolare geneticamente le linee cellulari somatiche hanno un grande potenziale per la ricerca di base e applicata. Qui presentiamo due approcci per la produzione e lo screening generati da CRISPR / Cas9 nelle linee cellulari mammiferi, con e senza l'uso di marcatori selectable.

Selezione-dipendente e generazione indipendente di CRISPR / Cas9-mediata Gene Knockouts nelle cellule di mammiferi

Questo protocollo descrive in dettaglio la generazione di cellule staminali pluripotenti indotte privo di impronta (iPSCs) da cellule pancreatiche umane in condizioni prive di alimentatore, seguito da editing con CRISPR/Cas9 ribonucleoproteine e caratterizzazione del modificati cloni unicellulari.

Efficiente generazione e la modifica di IPSCs privo di alimentatore da cellule pancreatiche umane utilizzando il sistema di CRISPR-Cas9

Un protocollo per la veloce rottura CRISPR/Cas9-mediata del gene nel topo ed emopoietici umani primari è descritto in questo articolo. Cas9-sgRNA ribonucleoproteine vengono introdotti tramite elettroporazione con sgRNAs generato tramite trascrizione in vitro e commerciale Cas9. Alta efficienza di editing si ottiene con tempo limitato e costi finanziari.

Rottura del Gene altamente efficiente di cellule progenitrici ematopoietiche Murine ed umane di CRISPR/Cas9

Qui, presentiamo un protocollo per modificare geneticamente le cellule primarie o espanso umana naturale dell'assassino (NK) utilizzando Cas9 ribonucleoproteine (Cas9/RNP). Utilizzando questo protocollo, abbiamo generato le cellule NK umane carenti per trasformare il recettore del fattore di crescita – b 2 (TGFBR2) e ipoxantina fosforibosiltransferasi 1 (HPRT1).

Generazione di cellule Knock-out NK umano primario ed espansa utilizzando Cas9 ribonucleoproteine

Presentato qui è un protocollo per l'efficiente editing genico mediato da ribonucleoproteina CRISPR/Cas9 nelle cellule dei mammiferi utilizzando l'elettroporazione dei tubi.

CRISPR/Cas9 Ribonucleoproteina-mediato Precision Gene Editing da Elettroporazione Tube

Il metodo presentato descrive la generazione di un knockout genico mediato da CRISPR nella linea H9 di cellule staminali embrionali umane (hESC), che esprime stabilmente sgRNA che hanno come bersaglio il gene L2HGDH utilizzando un sistema di rilascio genico lentivirale mediato altamente efficiente.

Delezione genica mediata da CRISPR-Cas9 in cellule staminali pluripotenti umane coltivate in condizioni prive di alimentatore

Qui, delineiamo un protocollo completo per la generazione e l'utilizzo di organoidi di glioblastoma ingegnerizzati in laboratorio (LEGO) per studiare le dipendenze genotipo-fenotipo e selezionare potenziali farmaci per il trattamento del glioblastoma.

Coltura di organoidi di glioblastoma ingegnerizzata in laboratorio e screening farmacologico

Viene fornito un protocollo dettagliato per l'utilizzo della tecnologia CRISPR/Cas9 per ottenere un knock-in mirato altamente efficiente di grandi costrutti multicistronici in cellule T umane primarie attraverso la via di riparazione del DNA HMEJ (omologia-mediata da end joining). Le cellule T ingegnerizzate con questo protocollo adattabile alle cGMP mantengono un'eccellente espansione cellulare, citotossicità e produzione di citochine.

Ingegnerizzazione non virale di cellule T umane primarie tramite integrazione mirata di giunzione terminale mediata da omologia di grandi modelli di DNA

Impara a modificare geneticamente le cellule THP-1 con CRISPR-Cas9 per studiare la resistenza dei macrofagi all'infezione da HIV. Questo protocollo include l'elettroporazione e l'isolamento di cloni a singola cellula.

Knockout genico mediato da CRISPR-Cas9 basato sull'elettroporazione in cellule THP-1 e isolamento di cloni a singola cellula

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