We wish to thank Dr. Daniel Gitler for providing us with the pAd Delta 5 Helper plasmid. This work was funded by the Israel Science Foundation (ISF, 700/16) (to ME), the United States-Israel Binational Science Foundation (BSF, 2017323) (to ME and MS), the Israel Cancer Association (ICA, 20170024) (to ME), the Israel Cancer Research Foundation (ICRF, 17-1693-RCDA) (to ME), and the Concern Foundation (#7895) (to ME). Fellowship: the Alon fellowship to ME and BGU Kreitman fellowships to SJ and MP.

This study was approved by the Ben Gurion University of the Negev Animal Care and Use Committee. Animal experiments were approved by the IACUC (IL.80-12-2015 and IL.29-05-2018(E)). All aspects of animal testing, housing, and environmental conditions used in this study were in compliance with The Guide for the Care and Use of Laboratory Animals13.
1. Adeno-associated virus production
<ol>
	<li>Day 1: Cell culture
	<ol>
		<li>Seed 4 x 106 HEK293T cells per 14...

<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. 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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=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. 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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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Several methods have previously been used to transform primary cells in culture with variable degrees of success25,26,27,28. Most of these methods use mouse fibroblast cells for transformation14,17,18,19 or use carcinogens such as 4-nitroquinoline-1-oxide (4-NQO)<sup ...

HNC is a common malignancy worldwide1. Modeling the genesis of HNC neoplasia is currently at a scientific turning point2. While many genetic mutations have been identified in HNC2,3,4 (e.g., TP53, PIK3CA, NOTCH1, FAT1, and RAS) the specific combinations of genomic alterations required in concert to induce HNC remain unclear.
The current use of human HNC cell lines has significantly helped to elucidate the mechanisms associated with pathogenesis and treatment3. However, the study of human cell lines in immunocompromised murine systems has its limitations, because these systems do not address the in vivo neoplastic process, the role of specific gene mutations, and treatment responses in an immune microenvironment. Hence, the development and establishment of murine cell lines with specific genetic alterations are of primary importance to study how different genes contribute to the transformation process and to test novel molecule-based therapies in immunocompetent mice.
Gene function studies in biomedical research have been significantly affected by advances in DNA editing technologies, by introducing double-strand breaks (DSBs), for example5. These technologies, including the use of zinc finger nucleases, transcription activator-like effector nucleases, and clustered regulatory interspaced short palindromic repeats (CRISPR/Cas9), allow for the manipulation of any gene of interest at its locus. The latest CRISPR/Cas9 systems is comprised of a guide RNA (gRNA) that directs the Cas9 nuclease to generate a DSB at a specific site in the genome. This system has gained extensive recognition in modifying endogenous genes in any cell or target tissue, even in the most traditionally difficult-to-treat organisms5. It has multiple advantages over other systems due to its simplicity, speed, and efficiency.
In oncology, CRISPR/CAS9 technology has fulfilled the need to effectively mimic cancer cells. To establish this system in HNC, we manipulated the potent KRAS oncogene and two important tumor suppressor genes, APC and p536. According to the GENIE database7, this combination is rare in HNC. RAS mutations (HRAS, NRAS, and KRAS) are present in only ~7% of all HNC populations. These tumors tend to be resistant to therapy8,9.
Delivery of Cas9 and its gRNA is achieved through viral transduction using either AAVs or lentiviruses. Recombinant AAV is often the preferred method for delivering genes to target cells owing to its high titer, mild immune response, ability to transduce a broad range of cells, and overall safety. Using an AAV system, various tissue-specific mouse cell lines have been generated, and new cell lines are still being developed10,11,12. However, an efficient genomic editing system that can generate murine HNC cell line models cells remains to be developed. In this study, we sought to develop an in vitro AAV-Cas9-based system for transforming primary murine tongue cells into a tumorigenic state. This unique CRISPR/Cas9 transformation protocol and the established tumor cell line can be used to better understand the biology of HNC induced by a diversity of genomic alterations.

<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

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by introducing specific mutations in oncogenes and tumor suppressor genes, using clustered regulatory interspaced short palindromic repeat (CRISPR)-based genome editing technology in mice. This technology allows us to accurately mimic the genetic changes that occur in human cancers using mice. By genetically transforming murine primary cells, we can better study cancer development, progression, treatment, and diagnosis. In this study, we used Cre-inducible Cas9 mouse tongue epithelial cells to enable genome editing using adeno-associated virus (AAV) in vitro. Specifically, by altering KRAS, p53, and APC in normal tongue epithelial cells, we generated a murine head and neck cancer (HNC) cell line in vitro,which is tumorigenic in syngeneic mice. The method presented here describes in detail how to generate HNC cell lines with specific genomic alterations and explains their suitability for predicting tumor progression in syngeneic mice. We envision that this promising method will be informative and useful to study tumor biology and therapy of HNC.

Using the AAV system to transform normal Cas9 cells 
Figure 1 provides a detailed vector map of the AAV transgene plasmid used in this study. Figure 2 outlines the design and working of the AAV-Cas9 based-system. To produce viral particles, the HEK293T cells were transfected with the AAV transgene vector and other viral packaging vectors using the PEI transfection method. After transfection, the virus-contai...

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

Development of murine models with specific genes mutated in head and neck cancer patients is required for understanding of neoplasia. Here, we present a protocol for in vitro transformation of primary murine tongue cells using an adeno-associated virus-Cas9 system to generate murine HNC cell lines with specific genomic alterations.

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

This protocol enabled the development of head and neck cancer models with specific genomic operation, significantly impacting our understanding of the role of specific gene mutations in the process of neoplasia. The main advantage of this technique is the ease with which the cell line can be developed from virtually any organ. Demonstrating the procedure will be Manu Prasad a grad student from my laboratory.Begin by using surgical scissors to harvest the tongue from a six week old male or female B6 transgenic mouse. Use a scalpel to mince the tissue into very small fragments and collect the pieces into a 15 milliliter tube containing 4.5 milliliters of RPMI plain medium without serum. Add 200 microliters of triple enzyme mix to the tube and incubate the sample at 37 degrees Celsius for 30 minutes tapping the tube every 10 minutes to enhance the enzymatic dissociation of the tissue.At the end of the incubation, stop the reaction with 5%FBS in PBS and filter the cell suspension through a 70 micro meter mesh strainer to separate the cells from the larger tissue fragments. Collect the cells by centrifugation and re-suspend the pellet in three milliliters of complete medium. Then plate the cells in three milliliters of complete medium per 16 millimeter dish.Transfer cell aggregates retained on top of the filter to a separate 16 millimeter culture dish and add three milliliters of complete medium. Keep both dishes in the incubator until distinct cell colonies are formed. After one week of culture, microscopically examine the primary cell cultures for the presence of fibroblast contamination, treating the cells with 25%trypsin in 02%EDTA at 37 degrees Celsius for one minute, and remove any fibroblasts.On day 10 of culture use 25%trypsin in 02%EDTA for three to five minutes at 37 degrees Celsius to harvest the cells from the cell suspension or cell aggregate cultures and see two times 10 to the fifth primary cells in two milliliters of medium per well into each well of the 6-well plate. The next day transduce the cells with one times 10 to the 12 viral genome per milliliter and incubate the cells in the viral particle-containing medium for 48 hours at 37 degrees Celsius. At the end of the incubation, replace the culture supernatants with two milliliters of fresh complete medium per well and return the cells to the cell culture incubator.After two weeks of culture expansion, seed the cells for validation and in vivo tumorigenic experiments. Using the AAV CAS9 vector, one times 10 to the 12 viral genomes per milliliter of virus have been determined to efficiently transform primary meth cells to tumorigenic maps using the protocol as demonstrated. The transduced cells Express Cre, CAS9 and Green Fluorescent Protein.Injection of five times 10 to the fifth primary AAV transduced maps into the tongue, lip and skin of NOD SCID mice, results in tumor formation in these animals unlike the injection of primary maps, which do not induce tumors. The tumorigenic transformation of primary tongue epithelial cells can be confirmed using immunofluorescence and western blotting. Genomic analysis by deep sequencing of DNA, isolated from the transformed cells, confirms the effective gene editing and frameshift of the tumor protein 53 and APC genes by the AAV CAS9 system.In addition, the transformed tongue cells efficiently form tumors in wild type C57 black six mice. Immunohistochemical analysis of the neoplastic tumor cells reveals cytoplasmic expression of E-Cadherin and Keratin 14, both epithelial tissue markers. When attempting this procedure, always remember that too much mincing or too much enzymatic reaction lead to reduced viability of the cells, leading to reduced virus transaction efficiency.Using this procedure, the cell lines can be genetically modified to gain insight into tumorigenic and metastatic potential of cancer cells from any tissue of interest.

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Research

JoVE Journal

Cancer Research

7.8K visualizações.  Ben-Gurion University of the Negev. Este protocolo possibilitou o desenvolvimento de modelos de câncer de cabeça e pescoço com operação genômica específica, impactando significativamente nossa compreensão do papel de mutações genéticas específicas no processo de neoplasia. A principal vantagem dessa técnica é a facilidade com que a linha celular pode ser desenvolvida a partir de praticamente qualquer órgão. Demonstrando o procedimento será Manu Prasad uma estudante de pós-graduação do meu laboratório.