Name: in vitro establishment of a genetically engineered murine head and neck cancer cell line using an adeno-associated virus-cas9 system
Uploaded: 2020-01-09T12:00:00
Description: Watch this Scientific Journal Video about In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System at JoVE.com

Vi vil gerne takke Dr. Daniel Gitler for at give os pAd Delta 5 Helper plasmid. Dette arbejde blev finansieret af Israel Science Foundation (ISF, 700/16) (til mig), de Forenede Stater-Israel binational Science Foundation (BSF, 2017323) (til mig og MS), Israel Cancer Association (ICA, 20170024) (for mig), Israel Cancer Research Foundation (ICRF, 17-1693-RCDA) (for mig), og concern Foundation (#7895) (for mig). Fællesskabet: Alon-stipendiet til mig og BGU Kreitman-stipendierne til SJ og MP.

Denne undersøgelse blev godkendt af Ben Gurion universitetet i Negev Animal Care og use udvalget. Dyreforsøg blev godkendt af IACUC (IL. 80-12-2015 og IL. 29-05-2018 (E)). Alle aspekter af dyreforsøg, boliger og miljøforhold, der anvendes i denne undersøgelse, var i overensstemmelse med vejledningen for pleje og brug af forsøgsdyr13.
1. adeno-associeret virus produktion
<ol><li>Dag 1: cellekultur<ol>
<li>Frø 4 x 106 HEK293T celler pr. 14,5 cm plade i...

<ol>
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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=CRISPR-Cas9+knockin+mice+for+genome+editing+and+cancer+modeling.">CRISPR-Cas9 knockin mice for genome editing and cancer modeling.</a> Cell. 159 (2), 440-455 (2014).</li><li>Zhang, Y., Chirmule, N., Gao, G. P., Wilson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40+ligand-dependent+activation+of+cytotoxic+T+lymphocytes+by+adeno-associated+virus+vectors+in+vivo:+role+of+immature+dendritic+cells.">CD40 ligand-dependent activation of cytotoxic T lymphocytes by adeno-associated virus vectors in vivo: role of immature dendritic cells.</a> Journal of Virology. 74 (17), 8003-8010 (2000).</li><li>Xiao, X., Li, J., Samulski, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+high-titer+recombinant+adeno-associated+virus+vectors+in+the+absence+of+helper+adenovirus.">Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus.</a> Journal of Virology. 72 (3), 2224-2232 (1998).</li><li>Auricchio, A., Hildinger, M., O'Connor, E., Gao, G. P., Wilson, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Highly+Infectious+and+Pure+Adeno-Associated+Virus+Type+2+Vectors+with+a+Single-Step+Gravity-Flow+Column.">Isolation of Highly Infectious and Pure Adeno-Associated Virus Type 2 Vectors with a Single-Step Gravity-Flow Column.</a> Human Gene Therapy. 12 (1), 71-76 (2001).</li><li>Lock, 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=Characterization+of+a+Recombinant+Adeno-Associated+Virus+Type+2+Reference+Standard+Material.">Characterization of a Recombinant Adeno-Associated Virus Type 2 Reference Standard Material.</a> Human Gene Therapy. 21 (10), 1273 (2010).</li><li>Challis, R. C., 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=Systemic+AAV+vectors+for+widespread+and+targeted+gene+delivery+in+rodents.">Systemic AAV vectors for widespread and targeted gene delivery in rodents.</a> Nature Protocols. 14 (2), 379-414 (2019).</li><li>Rabinowitz, J. E., 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=Cross-packaging+of+a+single+adeno-associated+virus+(AAV)+type+2+vector+genome+into+multiple+AAV+serotypes+enables+transduction+with+broad+specificity.">Cross-packaging of a single adeno-associated virus (AAV) type 2 vector genome into multiple AAV serotypes enables transduction with broad specificity.</a> Journal of Virology. 76 (2), 791-801 (2002).</li><li>Cheng, D. 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=Memorial+Sloan+Kettering-Integrated+Mutation+Profiling+of+Actionable+Cancer+Targets+(MSK-IMPACT).">Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT).</a> The Journal of Molecular Diagnostics. 17 (3), 251-264 (2015).</li><li>Sun, H., Taneja, R. <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+Transformation+and+Tumorigenicity+Using+Mouse+Embryonic+Fibroblast+Cells.">Analysis of Transformation and Tumorigenicity Using Mouse Embryonic Fibroblast Cells.</a> Cancer Genomics and Proteomics. 383, 303-310 (2007).</li><li>Durkin, M., Qian, X., Popescu, N., Lowy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Mouse+Embryo+Fibroblasts.">Isolation of Mouse Embryo Fibroblasts.</a> BIO-PROTOCOL. 3 (18), (2013).</li><li>Jozefczuk, J., Drews, K., Adjaye, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Mouse+Embryonic+Fibroblast+Cells+Suitable+for+Culturing+Human+Embryonic+and+Induced+Pluripotent+Stem+Cells.">Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells.</a> Journal of Visualized Experiments. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+transformation+of+mouse+embryo+fibroblasts+in+the+absence+of+activator+E2Fs.">Cellular transformation of mouse embryo fibroblasts in the absence of activator E2Fs.</a> Journal of Virology. 89 (9), 5124-5133 (2015).</li><li>Leong, H., Blewitt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrovirus+Mediated+Malignant+Transformation+of+Mouse+Embryonic+Fibroblasts.">Retrovirus Mediated Malignant Transformation of Mouse Embryonic Fibroblasts.</a> BIO-PROTOCOL. 3 (15), (2013).</li><li>Parikh, N., Nagarajan, P., Sei-ichi, M., Sinha, S., Garrett-Sinha, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+an+immortalized+oral+keratinocyte+cell+line+of+mouse+origin.">Isolation and characterization of an immortalized oral keratinocyte cell line of mouse origin.</a> Archives of Oral Biology. 53 (11), 1091-1100 (2008).</li><li>Badarni, 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=Repression+of+AXL+expression+by+AP-1/JNK+blockage+overcomes+resistance+to+PI3Ka+therapy.">Repression of AXL expression by AP-1/JNK blockage overcomes resistance to PI3Ka therapy.</a> JCI Insight. 5, 125341 (2019).</li><li>Chen, Y. F., 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=Establishing+of+mouse+oral+carcinoma+cell+lines+derived+from+transgenic+mice+and+their+use+as+syngeneic+tumorigenesis+models.">Establishing of mouse oral carcinoma cell lines derived from transgenic mice and their use as syngeneic tumorigenesis models.</a> BMC Cancer. 19 (1), 281 (2019).</li><li>Hoover, A. 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Flere metoder har tidligere været anvendt til at omdanne primære celler i kultur med varierende grader af succes25,26,27,28. De fleste af disse metoder bruger mus fibroblast celler til omdannelse14,17,18,19 eller bruge kræftfremkaldende stoffer såsom 4-nitroquinol...

HNC er en almindelig malignitet på verdensplan1. Modellering tilblivelsen af HNC neoplasi er i øjeblikket på et videnskabeligt vendepunkt2. Mens mange genetiske mutationer er blevet identificeret i HNC2,3,4 (f. eks. TP53, PIK3CA, NOTCH1, FAT1 og RAS), er de specifikke kombinationer af genomændringer, der kræves i koncert for at inducere HNC, stadig uklare.
Den nuværende brug af humane HNC cellelinjer har bidraget væsentligt til at belyse mekanismerne i forbindelse med patogenese og behandling3. Men studiet af humane cellelinjer i immunkompromitterede murine systemer har sine begrænsninger, fordi disse systemer ikke adresserer in vivo neoplastisk proces, rollen af specifikke genmutationer og behandlingsrespons i et immun mikromiljø. Derfor er udvikling og etablering af murine cellelinjer med specifikke genetiske ændringer af afgørende betydning for at undersøge, hvordan forskellige gener bidrager til transformationsprocessen, og for at afprøve nye molekyle baserede terapier hos immunkompetente mus.
Genfunktions undersøgelser i biomedicinsk forskning er blevet væsentligt påvirket af fremskridt inden for DNA-redigeringsteknologier ved at indføre dobbelt strengene pauser (DSBs), for eksempel5. Disse teknologier, herunder brugen af zink finger nucleaser, transkriptionsaktivator-lignende Effector nucleaser, og grupperet regulerende hinanden korte palindromiske gentagelser (crispr/Cas9), giver mulighed for manipulation af ethvert gen af interesse på sin locus. De seneste CRISPR/Cas9-systemer består af en guide RNA (gRNA), der dirigerer Cas9 nuclease til at generere en DSB på et bestemt sted i genomet. Dette system har opnået stor anerkendelse i at modificere endogene gener i enhver celle eller målvæv, selv i de mest traditionelt vanskelige at behandle organismer5. Det har flere fordele i forhold til andre systemer på grund af sin enkelhed, hastighed og effektivitet.
I onkologi har CRISPR/CAS9-teknologien opfyldt behovet for effektivt at efterligne kræftceller. For at etablere dette system i HNC, vi manipulerede den potente KRAS onkogen og to vigtige tumor suppressor gener, APC og P536. Ifølge GENIE database7, denne kombination er sjælden i HNC. RAS-mutationer (HRAS, NTM og KRAS) er kun til stede i ~ 7% af alle HNC-populationer. Disse tumorer tendens til at være modstandsdygtig over for terapi8,9.
Levering af Cas9 og dens gRNA opnås gennem viral transduktion ved hjælp af enten AAVs eller lentivira. Rekombinant AAV er ofte den foretrukne metode til at levere gener til at målrette celler på grund af sin høje titer, mild immunrespons, evne til at overføre en bred vifte af celler, og generelle sikkerhed. Ved hjælp af et AAV-system, forskellige vævsspecifikke muse cellelinjer er blevet genereret, og nye cellelinjer er stadig ved at blive udviklet10,11,12. Men, et effektivt genomisk redigeringssystem, der kan generere murine HNC cellelinje modeller celler mangler at blive udviklet. I denne undersøgelse, vi søgte at udvikle et in vitro AAV-Cas9-baseret system til omdannelse af primære murine tunge celler i en tumorigent tilstand. Denne unikke CRISPR/Cas9 transformation protokol og den etablerede tumor cellelinje kan bruges til bedre at forstå biologi HNC induceret af en mangfoldighed af genomiske ændringer.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti mouse HRP</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-035-146</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti rabbit HRP</td>
			<td>Jackson ImmunoResearch</td>
			<td>711-035-152</td>
			<td></td>
		</tr>
		<tr>
			<td>Cas9 Mouse mAb</td>
			<td>Cell Signaling Technology</td>
			<td>14697</td>
			<td></td>
		</tr>
		<tr>
			<td>Cre</td>
			<td>BioLegend</td>
			<td>900901</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3-AffiniPure Goat Anti-Mouse IgG</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-165-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy-AffiniPure Goat Anti-Rabbit IgG</td>
			<td>Jackson ImmunoResearch</td>
			<td>111-165-144</td>
			<td></td>
		</tr>
		<tr>
			<td>GFP</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-9996</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospho-p44/42 MAPK (Erk1/2)</td>
			<td>Cell Signaling Technology</td>
			<td>4370</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit monoclonal anti E cadherin</td>
			<td>Cell Signaling Technology</td>
			<td>3195S</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit monoclonal anti-KRT 14</td>
			<td>Abcam</td>
			<td>AB-ab181595</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946; actin</td>
			<td>MP Biomedicals</td>
			<td>691001</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946; catenin</td>
			<td>Cell Signaling Technology</td>
			<td>9582S</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell lines</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEK93T</td>
			<td>ATCC</td>
			<td><a href="https://www.atcc.org/en/Products/All/CRL-3216.aspx" target="_blank">CRL-3216</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Culture Media, Chemicals and Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bradford Reagent</td>
			<td>Bio-Rad</td>
			<td>30015484</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Amresco</td>
			<td>0332-TAM-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI fluoromount</td>
			<td>Southern Biotech</td>
			<td>0100-20</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Biological Industries Israel Beit-Haemek Ltd.</td>
			<td>01-055-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL (Westar Supernova and Westar Nova 2.0)</td>
			<td>Cyanagen</td>
			<td>XLS3.0100 and XLS071.0250</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Biological Industries Israel Beit-Haemek Ltd.</td>
			<td>04-127-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Sigma</td>
			<td>H6648</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin - Agarose</td>
			<td>Sigma</td>
			<td>H6508</td>
			<td></td>
		</tr>
		<tr>
			<td>Isolate II Genomic DNA Kit</td>
			<td>Bioline</td>
			<td>BIO-52066</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Panreac AppliChem</td>
			<td>300283</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Bio Lab Ltd</td>
			<td>1903059100</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Biological Industries Israel Beit-Haemek Ltd.</td>
			<td>02-023-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>PEI</td>
			<td>Polysciences</td>
			<td>23966-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen Strep Solution</td>
			<td>Biological Industries Israel Beit-Haemek Ltd.</td>
			<td>03-031-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA</td>
			<td>Santa Cruz Biotechnology</td>
			<td>30525-89-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphatase inhibitor cocktail</td>
			<td>Biotool</td>
			<td>B15001A/B</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>MilliporeSigma</td>
			<td>P2714-1BTL</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris buffer</td>
			<td>MERCK Millipore</td>
			<td>648311-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Enzymes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Benzonase</td>
			<td>Sigma</td>
			<td>E1014</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Thermo Fisher Scientific</td>
			<td>17104019</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse</td>
			<td>Thermo Fisher Scientific</td>
			<td>18047019</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>MilliporeSigma</td>
			<td>H3506</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Biological Industries Israel Beit-Haemek Ltd.</td>
			<td>03-050-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass wares</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slips</td>
			<td>Bar Naor</td>
			<td>BNCB00130RA1</td>
			<td></td>
		</tr>
		<tr>
			<td>Slides</td>
			<td>Bar Naor</td>
			<td>BN9308C</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6</td>
			<td>Envigo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B6;129-Gt(ROSA)26Sortm1(CAG-cas9*,-EGFP)Fezh/J</td>
			<td>Jackson labs</td>
			<td>24857</td>
			<td></td>
		</tr>
		<tr>
			<td>NOD.CB17-Prkdc-scid/NCr Hsd (Nod.Scid)</td>
			<td>Envigo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AAV pCM109 EFS Cre sg APC sg Kras sg P53- Kras HDR</td>
			<td>Broad Institute of MIT</td>
			<td></td>
			<td>Kind gift from Dr Randall J Platt and Dr. Joseph Rosenbluh, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA</td>
		</tr>
		<tr>
			<td>AAV 2/9 capsid vector</td>
			<td>Addgene</td>
			<td>112865</td>
			<td></td>
		</tr>
		<tr>
			<td>pAD Delta F5 helper</td>
			<td>Ben Gurion University of the Negev</td>
			<td></td>
			<td>Provided by Dr Daniel Gitler, Department of Physiology and Cell Biology, Faculty of Health Sciences, and Zlotowski Center for Neuroscience, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel.</td>
		</tr>
		<tr>
			<td>Plastic wares</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon-ULTRA filter 100 KDa</td>
			<td>Millipore</td>
			<td>UFC910024</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#181;m sterile filters, 4 mm</td>
			<td>Millex</td>
			<td>SLGV004SL</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 &#181;m sterile filters, 13 mm</td>
			<td>Millex</td>
			<td>SLHV013SL</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture plates</td>
			<td>Greiner Bio-One</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tubes</td>
			<td>Greiner Bio-One</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro establishment of a genetically engineered murine head and neck cancer cell line using an adeno-associated virus-cas9 system

Brugen af primære normale epitelceller gør det muligt at reproducerbart inducere genomiske ændringer, der kræves for cellulære transformation ved at indføre specifikke mutationer i onkogener og tumor suppressor gener, ved hjælp af klyngeopdelte regulerende hinanden kort palindromiske REPEAT (crispr)-baseret genom redigerings teknologi i mus. Denne teknologi giver os mulighed for nøjagtigt at efterligne de genetiske ændringer, der opstår i menneskelige kræftformer ved hjælp af mus. Ved genetisk omdanne murine primære celler, vi kan bedre studere kræft udvikling, progression, behandling, og diagnose. I denne undersøgelse, vi brugte CRE-inducerbar Cas9 mus tungen epitelceller til at muliggøre genomredigering ved hjælp af adeno-associeret virus (AAV) in vitro. Specifikt, ved at ændre KRAS, P53 og APC i normale tunge epitelceller, vi genereret en murine hoved og halscancer (HNC) cellelinje in vitro, som er tumorigent i syngeneiske mus. Den metode, der præsenteres her, beskriver i detaljer, hvordan man genererer HNC cellelinjer med specifikke genomændringer og forklarer deres egnethed til at forudsige tumorprogression i syngeneiske mus. Vi forestiller, at denne lovende metode vil være informativ og nyttig til at studere tumor biologi og terapi af HNC.

Brug af AAV-systemet til at omdanne normale Cas9 celler Figur 1 indeholder et detaljeret vektor kort over AAV transgene plasmid, der anvendes i dette studie. Figur 2 skitserer udformningen og arbejdet af AAV-Cas9 baseret-system. For at fremstille virale partikler blev de HEK293T celler transficeret med AAV transgen vektor og andre virale emballage vektorer ved hjælp af Pei transfektering metode. Efter transfe...

Watch this Scientific Journal Video about In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System at JoVE.com

In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System

Udvikling af murine modeller med specifikke gener muteret i hoved og halscancer patienter er nødvendig for forståelsen af neoplasi. Her præsenterer vi en protokol for in vitro-omdannelse af primære murine tunge celler ved hjælp af en adeno-associeret virus-Cas9 system til at generere murine HNC cellelinjer med specifikke genomiske ændringer.

In vitro etablering af en genetisk manipuleret murine hoved og hals Cancer cellelinje ved hjælp af en adeno-associeret virus-Cas9 system

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by ...

Denne protokol muliggjorde udviklingen af hoved- og halskræftmodeller med specifik genomisk drift, hvilket i væsentlig grad påvirkede vores forståelse af specifikke genmutationers rolle i processen med neoplasi. Den største fordel ved denne teknik er den lethed, hvormed cellelinjen kan udvikles fra stort set ethvert organ. Demonstration af proceduren vil være Manu Prasad en grad studerende fra mit laboratorium.Begynd med at bruge kirurgisk saks til at høste tungen fra en seks uger gammel mandlig eller kvindelig B6 transgen mus. Brug en skalpel til at hakke vævet i meget små fragmenter og opsaml stykkerne i et 15 milliliterrør indeholdende 4,5 milliliter RPMI almindeligt medium uden serum. Der tilsættes 200 mikroliter tredobbelt enzymblanding til røret, og prøven inkuberes ved 37 grader Celsius i 30 minutter og aflyse røret hvert 10.Ved inkubationens afslutning stoppes reaktionen med 5%FBS i PBS, og cellesuspensionen filtreres gennem en 70 mikrometermaskes si for at adskille cellerne fra de større vævsfragmenter. Cellerne opsamls ved centrifugering, og pellets skal igen suspenderes i tre milliliter komplet medium. Derefter plade cellerne i tre milliliter komplet medium pr 16 millimeter skål.Overfør celle aggregater bevaret oven på filteret til en separat 16 millimeter kultur skål og tilsæt tre milliliter komplet medium. Hold begge retter i inkubatoren, indtil der dannes forskellige cellekolonier. Efter en uges kultur undersøger mikroskopisk de primære cellekulturer for tilstedeværelsen af fibroblastforurening, behandling af cellerne med 25 % trypsin i 02%EDTA ved 37 grader Celsius i et minut, og fjern eventuelle fibroblaster.På dag 10 af kulturen bruge 25% trypsin i 02% EDTA i tre til fem minutter ved 37 grader Celsius at høste cellerne fra celle suspension eller celle aggregat kulturer og se to gange 10 til den femte primære celler i to milliliter medium pr godt ind i hver brønd af 6-brønd plade. Den næste dag transduce cellerne med en gange 10 til 12 virale genom pr milliliter og inkubere cellerne i den virale partikelholdige medium i 48 timer ved 37 grader Celsius. Ved slutningen af inkubationen erstattes kultursupernatanterne med to milliliter frisk komplet medium pr. brønd, og cellerne returneres til cellekulturkubvøsen.Efter to ugers kultur ekspansion, frø cellerne til validering og in vivo tumorigenic eksperimenter. Ved hjælp af AAV CAS9 vektor, en gange 10 til de 12 virale genomer per milliliter af virus er blevet fast besluttet på effektivt at omdanne primære meth celler til tumorigenic kort ved hjælp af protokollen som påvist. De transduced celler Express Cre, CAS9 og Green Fluorescerende Protein.Injektion af fem gange 10 til den femte primære AAV transduced kort i tungen, læbe og hud af NOD SCID mus, resulterer i tumordannelse i disse dyr i modsætning til injektion af primære kort, som ikke fremkalder tumorer. Den tumorigenic transformation af primære tungen epitelceller kan bekræftes ved hjælp af immunfluorescens og vestlige blotting. Genomisk analyse ved dyb sekventering af DNA, isoleret fra de transformerede celler, bekræfter den effektive genredigering og frameshift af tumorproteinet 53 og APC-gener af AAV CAS9-systemet.Desuden danner de transformerede tungeceller effektivt tumorer i vild type C57 sorte seks mus. Immunohistokemisk analyse af de neoplastiske tumorceller afslører cytoplasmatisk udtryk for E-Cadherin og Keratin 14, begge epitelvæv markører. Når du forsøger denne procedure, altid huske, at for meget hakning eller for meget enzymatisk reaktion føre til reduceret levedygtighed af cellerne, hvilket fører til reduceret virus transaktion effektivitet.Ved hjælp af denne procedure, cellelinjer kan genetisk modificeret for at få indsigt i tumorigenic og metastatisk potentiale af kræftceller fra ethvert væv af interesse.

Research

JoVE Journal

Cancer Research

A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma

En model for perineurale invasion i hoved og hals pladecellecarcinom

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, ...

Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinoma Samples

Four-Color Fluorescence Immunohistochemistry af T-Cell Subpopulations i Archival Formalin-Fixed, Paraffin-Embedded Human Oropharyngeal Squamous Cell Carcinoma Samples

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their ...

A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer

En genetisk manipuleret musemodel af sporadisk kolorektal cancer

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations ...

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Etablering af en lunge kolonisering Assay for Cirkulerende Tumor Cell visualisering i lungevæv

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

Terapi test i en klumpformet-baseret 3D celle kultur Model for hoved og hals planocellulært karcinom

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with ...

Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

Berigelse og karakterisering af Tumor immun- og ikke-immune Microenvironments i etablerede subkutane Murine tumorer

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for ...

Live Cell Imaging of the TGF-	&beta;/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System

Live Cell Imaging of the TGF-	ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â²/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System

Live celle billeddannelse af den TGF-β/Smad3 Signaling Pathway In Vitro og In Vivo ved hjælp af en Adenovirus Reporter System

Transforming Growth Factor &#946; (TGF-&#946;) signaling regulates many important functions required for cellular homeostasis and is commonly found ...

Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells

Vurdering cellelevedygtighed og død i 3D Spheroid kulturer af kræftceller

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

Etablering af gastrisk Cancer patient afledte xenograft-modeller og primære cellelinjer

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

In Vitro Assay to Study Tumor-macrophage Interaction

In vitro-analyse for at studere tumor-makrofag interaktion

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...