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 14.5 cm plate in 15 mL of DMEM. Prepare 10 plates for transfection with polyethylenimine (PEI) (1 &#181;g/&#181;L).</li>
	</ol>
	</li>
	<li>Day 2: Transfection of HEK293T cells using PEI
	<ol>
		<li>Remove media and refeed with warm DMEM 1 h before the transfection.</li>
		<li>Prewarm transfection reagents and DMEM.</li>
		<li>Use 10 &#181;g of the plasmid of interest containing AAV ITR (AAV pCM109 EFS Cre sg APC sg KRAS sg P53- KRAS HDR), 10 &#181;g of the AAV 2/9n capsid plasmid (with the rep gene of AAV2 and the cap gene of AAV9, and 10 &#181;g of the helper plasmid (pAdDelta F5 helper) per plate (See Table of Materials). 
		NOTE: A new generation of helper plasmid - pAdDeltaF614,15,16 that can also be used for AAV production is available.</li>
		<li>Mix the plasmids in 1 mL of plain DMEM per plate. Then add 90 &#181;L of polyethylenimine (total plasmid: PEI concentration = 1:3) to the plasmid mix and vortex briefly.</li>
		<li>Incubate the PEI-plasmid DNA mix for 20 min at room temperature (RT).</li>
		<li>Add the mix dropwise on top of the HEK293T cells and transfer the plates to the cell culture incubator at 37 &#176;C with 5% CO2 for 24 h.</li>
	</ol>
	</li>
	<li>Day 3: Medium change after transfection
	<ol>
		<li>Remove the medium completely and add 15 mL of fresh DMEM.</li>
	</ol>
	</li>
	<li>Day 5: Harvesting the virus
	<ol>
		<li>Prepare a dry ice/ethanol bath.</li>
		<li>Collect the cells with a cell scraper and transfer them into 50 mL tubes (2 plates per 50 mL tube).</li>
		<li>Spin tubes at 800 x g for 15 min at room temperature.</li>
		<li>Discard the supernatant from all the tubes. Add 0.5 mL of the lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH = 8.5) per plate (i.e., 5 mL for 10 plates) to the first tube only. Resuspend the cells and transfer the total volume to the next tube and continue until the last tube.</li>
		<li>Transfer the cell suspension to a fresh 50 mL tube. Wash the tubes with the same volume of lysis buffer (0.5 mL per plate) using the same transfer method as described in step 1.4.4.</li>
		<li>Subject the cell suspension to three rounds of ~10 min freeze/thaw cycles between a dry ice/ethanol bath and a 37 &#176;C water bath. Vortex briefly after each thawing.</li>
		<li>If the purification is carried out on the same day, set the equilibration and elution buffer (see Table 1 for the recipe) at RT.</li>
		<li>Add 50 units of benzonase per plate and incubate at 37 &#176;C for 1.5 h. 
		NOTE: Benzonase is used to digest residual nucleic acids from the host producer cells and the plasmid DNA present in the cell suspension.</li>
		<li>Spin the tubes at 3,000 x g for 15 min at 4 &#176;C.</li>
		<li>Collect the supernatant in a syringe using a 18 G steel needle and push the solution through a 0.45 &#181;m filter into a 15 mL tube to obtain the crude lysate.</li>
		<li>Store the crude lysate at 4 &#176;C for a few weeks until purification or continue purification.</li>
	</ol>
	</li>
</ol>
2. AAV purification
<ol>
	<li>Day 5 or later
	<ol>
		<li>Place the equilibration and elution buffer at RT.</li>
		<li>Wash chromatography columns with 10 mL of the equilibration buffer.</li>
		<li>Add 0.5 mL per plate of heparin-agarose to the column17 and then add the equilibration buffer (4x the volume of the heparin-agarose). Mix the solutions by inverting the column and let the agarose sediment.</li>
		<li>Elute the equilibration buffer from the column by gravity. 
		NOTE: Leave some equilibration buffer to prevent the agarose from drying out.</li>
		<li>Load the crude lysate onto the column and incubate for 2 h at 4 &#176;C with constant agitation. Bring the column into an upright position and allow the agarose to sediment.</li>
		<li>Elute the crude lysate by gravity.</li>
		<li>Wash the column with equilibration buffer (4x the volume of the heparin-agarose).</li>
		<li>Place a 100 kDa centrifugal protein filter below the column and elute the virus using an elution buffer (3x the volume of the heparin-agarose) into the filter. 
		NOTE: The elution buffer should not exceed 15 mL.</li>
		<li>Centrifuge the filter at 3,000 x g for ~30 min until less than 1 mL is left in the filter.</li>
		<li>Fill up the filter with PBS and centrifuge at 3,000 x g for ~30 min until less than 1 mL is left in the filter. Repeat this step 2x to remove all salts.</li>
		<li>Concentrate the virus solution in the filter by centrifugation at 3,000 x g to arrive at a volume as small as possible (less than 200 &#181;L).</li>
		<li>Aspirate the virus solution with a needle and syringe and push the solution through a 0.22 &#181;m filter into a tube.</li>
		<li>Make aliquots of concentrated viral particles for storage. 
		NOTE: The aliquots should be ~20 &#181;L for short-term storage at 4 &#176;C and ~5 &#181;L for long-term storage at -80 &#176;C.</li>
		<li>Determine the viral titer and viral transduction efficiency as described previously14,18,19,20.</li>
	</ol>
	</li>
</ol>
3. Isolation and culture of primary cells
<ol>
	<li>Day 1
	<ol>
		<li>Euthanize a 6-week-old male or female B6;129-Gt(ROSA)26Sortm1(CAG-cas9*,-EGFP)Fezh/J by CO2 asphyxiation or any other IACUC approved protocol. 
		NOTE: These CRISPR/Cas9 knockin mice have Cre recombinase-dependent expression of cas9 endonuclease and EGFP directed by a CAG promoter. The upstream Lox-Stop-Lox (LSL) sequence present in the genome of these mice prevent the expression of Cas9 and EGFP in the absence of Cre recombinase. When used in combination with single guide RNAs (sgRNAs) and a Cre source, they allow editing of single or multiple mouse genes in vivo or ex vivo14.</li>
		<li>Dissect the tongue from the euthanized mice using surgical scissors.</li>
		<li>Manually dissociate the tissue by mincing the tongue tissue into very small fragments using a scalpel. Collect the tissue fragments in a 15 mL tube containing 4.5ml of RPMI plain medium (with out serum).</li>
		<li>Add the triple enzyme mix (200 &#181;l) (see Table 1 for the recipe) to the tissue fragments.</li>
		<li>Incubate the tissue-enzyme mix at 37 &#176;C for 30 min and tap the tube every 10 min to enhance enzymatic dissociation of the tissue.</li>
		<li>Add 5% fetal bovine serum (FBS) containing HBSS/PBS to the tissue-enzyme mix to stop the enzyme action.</li>
		<li>Filter the above cell suspension through a sterile 70 &#181;m nylon mesh to separate the dispersed cells and larger tissue fragments.</li>
		<li>Wash the filtered cell suspension by centrifugation for 300 x g for 5 min in HBSS/PBS at RT.</li>
		<li>Resuspend the pellet in culture medium (10% FBS in RPMI/DMEM) and grow in 60 mm culture dishes until distinct cell colonies are formed.
		<ol>
			<li>Culture cell aggregates retained on top of the filter in a 60 mm culture dish containing 3 mL of complete media (10% FBS in RPMI/DMEM) until cell colonies are formed.</li>
		</ol>
		</li>
		<li>Microscopically examine the primary cells for the presence of fibroblast contamination after 1 week of culture. Treat the primary cells developed from aggregates and cell suspensions with 0.25 trypsin 0.02% EDTA solution at 37 &#176;C for 1 min to remove fibroblasts. 
		NOTE: Usually the primary culture from cell aggregates produces more colonies compared to single-cell suspensions. The cells from these colonies provide better transduction efficiency with AAV transduction.</li>
	</ol>
	</li>
</ol>
4. AAV transduction of primary cells
<ol>
	<li>Day 10
	<ol>
		<li>Seed 2 x 105 primary cells per well in 6 well plates in 2 mL of complete media (10% FBS in RPMI/DMEM).</li>
		<li>The next day, transduce the cells with 1012 viral genome/mL (1010 transducing units/mL) and incubate the cells in viral particle-containing media for 48 h at 37 &#176;C.</li>
		<li>Remove the viral particle-containing media and feed the AAV-transduced cells with complete media. 
		NOTE: Only cells that underwent transduction will express GFP and start to proliferate. Cells that did not undergo transduction will eventually die within 2 weeks.</li>
		<li>After 2 weeks of culture expansion, seed the cells for validation and the in vivo tumorigenic experiments. 
		NOTE: Genomic DNA fromAAV-transduced cells and normal primary cells from Cas9 mice were extracted for validating specific genome editing using standard protocols. Sequencing of the extracted DNA and analysis of the sequenced data (Table 2) were performed using a hybridization capture-based next-generation sequencing assay (e.g., MSK-IMPACT platform) as described previously21.</li>
	</ol>
	</li>
</ol>
5. Validating the transformation of normal cells to tumorigenic cells using immunofluorescence and western blotting
<ol>
	<li>Immunofluorescence
	<ol>
		<li>Seed 2 x 105 cells on 12 mm glass coverslips and place them in an incubator overnight.</li>
		<li>Fix cells in 4% paraformaldehyde in PBS (pH = 7.4) and process for immunofluorescence labeling.</li>
		<li>Incubate the fixed cells with the first primary antibody in 1% BSA or 1% serum in PBST in a humidified chamber for 1 h at RT or overnight at 4 &#176;C. The primary antibodies used are the rabbit monoclonal anti-KRT 14, rabbit monoclonal anti-E-cadherin antibody, and Cas9 mouse mAb.</li>
		<li>Remove the primary antibody solution from the coverslips by washing the cells 3x for 5 min with 1x PBS.</li>
		<li>Incubate the cells with Cy3 donkey anti-rabbit IgG and/or Cy3 goat anti-mouse IgG secondary antibodies in 5% BSA in PBST for 1 h at RT in the dark.</li>
		<li>Remove the secondary antibody solution from the coverslips and wash the cells 3x as described in step 5.1.4.</li>
		<li>Mount the coverslips with a drop of mounting medium containing DAPI (DAPI Fluoromount).</li>
		<li>Store in the dark at 4 &#176;C until the slides are imaged using fluorescence microscopy.</li>
	</ol>
	</li>
	<li>Western blotting
	<ol>
		<li>Seed 1 x 106 transformed cells in a 100 mm culture dish and place them in an incubator overnight.</li>
		<li>Wash and scrape the transformed cells into 200 &#181;l ice cold PBS.</li>
		<li>Centrifuge the tube containing the cell suspension for 10 min at 2,000 x g at 4 &#176;C to pellet the cells.</li>
		<li>Aspirate the supernatant and lyse the cells using lysis buffer (see Table 1) containing phosphatase inhibitor cocktails and a protease inhibitor for 10 min at 4 &#176;C. Centrifuge the lysates for 10 min at 10,000 x g and 4 &#176;C and collect the cleared lysates.</li>
		<li>Use a commercially available Bradford assay kit to determine the protein concentration following the manufacturer&#39;s protocol. Use 4x sample buffer (500 mM Tris pH = 6.8, 40% glycerol, 8% SDS, 20% H2O, 0.02% bromophenol blue) to adjust the protein samples to 0.5 or 1 &#181;g/&#181;L. Boil at 96 &#176;C for 5 min. 
		NOTE: The samples can be stored at -80 &#176;C or until a Western blot analysis is performed.</li>
		<li>Separate equal amounts of total lysate (30 &#181;g) by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Ensure that the dye reaches the bottom of the gel. Transfer the protein to the nylon membrane by semi-dry blotting at 25 V for 30 min.</li>
		<li>Pour 5% BSA in Tris-buffered saline (TBS)-0.1% Tween (TBST) over the membrane to cover it completely. Block the membrane for 1 h at RT. Incubate the mebrane with primary antibodies (anti-Cre, anti-Cas9, anti-GFP, anti-&#946; catenin, anti p53, anti-phospho-ERK, and anti-&#946; actin diluted in 5% BSA TBST) at 4 &#176;C overnight.</li>
		<li>After incubation, wash the membranes for 10 min with 1x TBST making sure that the solution covers the membrane completely. Perform this wash 3x, and then add horseradish peroxidase (HRP)-conjugated secondary antibody (diluted 1:10,000 in 5% BSA TBST) to the membrane. Incubate for 1 h at RT.</li>
		<li>After incubation, wash the membranes for 10 min with 1x TBST making sure that the solution covers the membrane completely. Perform the chemiluminescence imaging (see Table of Materials) to expose the bands, and capture images accordingly.</li>
	</ol>
	</li>
	<li>Validating the tumorigenic potential of transformed cells in immunocompetent mice 
	NOTE: Mice were maintained and treated according to the institutional guidelines of the Ben-Gurion University of the Negev. NOD.CB17-Prkdc-scid/NCr Hsd (NOD.SCID) and C57BL/6 mice were used for the in vivo studies. Mice were housed in air-filtered laminar flow cabinets with a 12 hour light/dark cycle and were fed food and water ad libitum.
	<ol>
		<li>Use 6-8 week-old female NOD.CB17-Prkdc-scid/NCr Hsd (NOD.SCID) and C57BL/6 mice for the study.</li>
		<li>Trypsinize the AAV-Cas9 transformed cells. Stop the trypsinization using DMEM prewarmed at 37 &#176;C and collect in a 50 mL tube.</li>
		<li>Centrifuge the tube at 800 x g for 10 min at room temperature. Discard the supernatant and resuspend the cell pellet in DMEM medium without FBS. Perform the centrifugation again and resuspend the cell pellet in 1x PBS. 
		NOTE: Do not keep the cells in 1x PBS for too long. Always keep the cell suspension on ice to prevent cell clumping.</li>
		<li>Use an automatic cell counter to count the cells and dilute the cells to the desired concentration (2.5 x 107 cells/mL) using 1x PBS. Generate tumors through a subcutaneous injection of the AAV-Cas9 transformed cell suspension in the right flank of each NOD.CB17-Prkdc-scid/NCr Hsd (NOD.SCID) mouse (2 x 106 cells/mouse). To generate an orthotropic model in syngeneic mice, inject 0.5 &#215; 106 primary cells or the AAV-Cas9 transformed cells into the tongue of C57BL/6 immunocompetent mice.</li>
		<li>Euthanize the animals 2 weeks postinjection by CO2 asphyxiation, and dissect the tumors from the euthanized mice for immunohistochemistry analysis.</li>
	</ol>
	</li>
</ol>

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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. (64), e3854 (2012).</li><li>Boehm, J. S., Hession, M. T., Bulmer, S. E., Hahn, W. C. <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+Human+and+Murine+Fibroblasts+without+Viral+Oncoproteins.">Transformation of Human and Murine Fibroblasts without Viral Oncoproteins.</a> Molecular and Cellular Biology. 25 (15), 6464 (2005).</li><li>Gupta, T., S&#225;enz Robles, T. M., Pipas, 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=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. 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=The+Role+of+Human+Papillomavirus+16+E6+in+Anchorage-Independent+and+Invasive+Growth+of+Mouse+Tonsil+Epithelium.">The Role of Human Papillomavirus 16 E6 in Anchorage-Independent and Invasive Growth of Mouse Tonsil Epithelium.</a> Archives of Otolaryngology-Head &amp; Neck Surgery. 133 (5), 495 (2007).</li><li>Smahel, 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=Metastatic+MHC+class+I-negative+mouse+cells+derived+by+transformation+with+human+papillomavirus+type+16.">Metastatic MHC class I-negative mouse cells derived by transformation with human papillomavirus type 16.</a> British Journal of Cancer. 84 (3), 374-380 (2001).</li><li>He, L., 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=Increased+Growth+of+a+Newly+Established+Mouse+Epithelial+Cell+Line+Transformed+with+HPV-16+E7+in+Diabetic+Mice.">Increased Growth of a Newly Established Mouse Epithelial Cell Line Transformed with HPV-16 E7 in Diabetic Mice.</a> PloS One. 11 (10), e0164490 (2016).</li></ol>

M.S. is on the Advisory Board of the Bioscience Institute, and Menarini Ricerche has received research funds from Puma Biotechnology, Daiichi-Sankio, Targimmune, Immunomedics, and Menarini Ricerche. M.S. is also a co-founder of Medendi Medical Travel, and in the past 2 years, has received honoraria from Menarini Ricerche and ADC Pharma. All other authors have nothing to disclose.

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><tbody><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</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>&amp;beta; actin</td><td>MP Biomedicals</td><td>691001</td><td></td></tr><tr><td>&amp;beta; catenin</td><td>Cell Signaling Technology</td><td>9582S</td><td></td></tr><tr><td>&lt;strong&gt;Cell lines&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>HEK93T</td><td>ATCC</td><td>&lt;a href=&quot;https://www.atcc.org/en/Products/All/CRL-3216.aspx&quot; target=&quot;_blank&quot;&gt;CRL-3216&lt;/a&gt;</td><td></td></tr><tr><td>&lt;strong&gt;Culture Media, Chemicals and Reagents&lt;/strong&gt;</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>MgCl&lt;sub&gt;2&lt;/sub&gt;</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>&lt;strong&gt;Enzymes&lt;/strong&gt;</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>&lt;strong&gt;Glass wares&lt;/strong&gt;</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>&lt;strong&gt;Mouse strains&lt;/strong&gt;</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)26Sor&lt;sup&gt;tm1(CAG-cas9*,-EGFP)Fezh&lt;/sup&gt;/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>&lt;strong&gt;Plasmids&lt;/strong&gt;</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>&lt;strong&gt;Plastic wares&lt;/strong&gt;</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 &amp;micro;m sterile filters, 4 mm</td><td>Millex</td><td>SLGV004SL</td><td></td></tr><tr><td>0.45 &amp;micro;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 ...

in-vitro-establishment-genetically-engineered-murine-head-neck-cancer

Research

JoVE Journal

Cancer Research

7.8K Views.  Ben-Gurion University of the Negev. Dit protocol maakte de ontwikkeling mogelijk van hoofd- en halskankermodellen met een specifieke genomische werking, wat een aanzienlijke invloed had op ons begrip van de rol van specifieke genmutaties in het proces van neoplasie. Het belangrijkste voordeel van deze techniek is het gemak waarmee de cellijn kan worden ontwikkeld vanuit vrijwel elk orgaan. Het aantonen van de procedure zal Manu Prasad een grad student uit mijn laboratorium.Begin met het gebruik van chirurgische schaar om...

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

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Medicine

Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration

With an increasing incidence of prostate cancer, identification of new tumor drivers or modulators is crucial. Genetically engineered mouse models (GEMM) for prostate cancer are hampered by tumor heterogeneity and its complex microevolution dynamics. Traditional prostate cancer mouse models include, amongst others, germline and conditional knockouts, transgenic expression of oncogenes, and xenograft models. Generation of de novo mutations in these models is complex, time-consuming, and costly. In addition, most of traditional models target the majority of the prostate epithelium, whereas human prostate cancer is well known to evolve as an isolated event in only a small subset of cells. Valuable models need to simulate not only prostate cancer initiation, but also progression to advanced disease.
Here we describe a method to target a few cells in the prostate epithelium by transducing cells by viral particles. The delivery of an engineered virus to the murine prostate allows alteration of gene expression in the prostate epithelia. Virus type and quantity will hereby define the number of targeted cells for gene alteration by transducing a few cells for cancer initiation and many cells for gene therapy. Through surgery-based injection in the anterior lobe, distal from the urinary track, the tumor in this model can expand without impairing the urinary function of the animal. Furthermore, by targeting only a subset of prostate epithelial cells the technique enables clonal expansion of the tumor, and therefore mimics human tumor initiation, progression, as well as invasion through the basal membrane.
This novel technique provides a powerful prostate cancer model with improved physiological relevance. Animal suffering is limited, and since no additional breeding is required, overall animal count is reduced. At the same time, analysis of new candidate genes and pathways is accelerated, which in turn is more cost efficient.

This protocol describes a newly established method for virus delivery to the murine prostate. Using either CRISPR/Cas9 technology, gene overexpression, or Cre recombinase delivery, the technique allows orthotopic alteration of gene expression and implements a novel mouse model for prostate cancer.

With an increasing incidence of prostate cancer, identification of new tumor drivers or modulators is crucial. Genetically engineered mouse models (GEMM) ...

Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland

Breast cancer is a heterogeneous disease, possibly due to complex interactions between different cells of origins and oncogenic events. Mouse models are instrumental in gaining insights into these complex processes. Although many mouse models have been developed to study contributions of various oncogenic events and cells of origin to breast tumorigenesis, these models are often not cell-type or organ specific or cannot induce the initiation of mammary tumorigenesis in a temporally controlled manner. Here we describe a protocol to generate a new type of breast cancer mouse models based on the intraductal injection of Cre-expressing adenovirus (Ad-Cre) into mouse mammary glands (MGs). Due to the direct injection of Ad-Cre into mammary ducts, this approach is MG specific, without any unwanted cancer induction in other organs. The intraductal injection procedure can be performed in mice at different stages of their MG development (thus, it permits temporal control of cancer induction, starting from ~3-4 weeks of age). The cell-type specificity can be achieved by using different cell-type-specific promoters to drive Cre expression in the adenoviral vector. We show that luminal and basal mammary epithelial cells (MECs) can be tightly targeted for Cre/loxP-based genetic manipulation via an intraductal injection of Ad-Cre under the control of the Keratin 8 or Keratin 5 promoter, respectively. By incorporating a conditional Cre reporter (e.g., Cre/loxP-inducible Rosa26-YFP reporter), we show that MECs targeted by Ad-Cre, and tumor cells derived from them, can be traced by following the reporter-positive cells after intraductal injection.

The goal of this protocol is to describe a new breast cancer modeling approach based on the intraductal injection of Cre-expressing adenovirus into mouse mammary glands. This approach allows both cell-type- and organ-specific manipulation of oncogenic events in a temporally controlled manner.

Breast cancer is a heterogeneous disease, possibly due to complex interactions between different cells of origins and oncogenic events. Mouse models are ...

In Vivo CRISPR/Cas9 Screening to Simultaneously Evaluate Gene Function in Mouse Skin and Oral Cavity

Genetically modified mouse models (GEMM) have been instrumental in assessing gene function, modeling human diseases, and serving as preclinical model to assess therapeutic avenues. However, their time-, labor- and cost-intensive nature limits their utility for systematic analysis of gene function. Recent advances in genome-editing technologies overcome those limitations and allow for the rapid generation of specific gene perturbations directly within specific mouse organs in a multiplexed and rapid manner. Here, we describe a CRISPR/Cas9-based method (Clustered Regularly Interspaced Short Palindromic Repeats) to generate thousands of gene knock-out clones within the epithelium of the skin and oral cavity of mice, and provide a protocol detailing the steps necessary to perform a direct&#160;in vivo&#160;CRISPR&#160;screen for tumor suppressor genes. This approach can be applied to other organs or other CRISPR/Cas9 technologies such as CRISPR-activation or CRISPR-inactivation to study the biological function of genes during tissue homeostasis or in various disease settings.

Here we describe a rapid and direct in vivo CRISPR/Cas9 screening methodology using ultrasound-guided in utero embryonic lentiviral injections to simultaneously assess functions of several genes in the skin and oral cavity of immunocompetent mice.

Genetically modified mouse models (GEMM) have been instrumental in assessing gene function, modeling human diseases, and serving as preclinical model to ...

Genetics

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites have been the gold standards for conditional or inducible gene targeting. To provide faster and more efficient experimental models, an ex vivo organoid culture system is developed using adenovirus Cre and normal urothelial cells carrying multiple loxP alleles of the tumor suppressors Trp53, Pten, and Rb1. Normal urothelial cells are enzymatically disassociated from four bladders of triple floxed mice (Trp53f/f: Ptenf/f: Rb1f/f). The urothelial cells are transduced ex vivo with adenovirus-Cre driven by a CMV promoter (Ad5CMVCre). The transduced bladder organoids are cultured, propagated, and characterized in vitro and in vivo. PCR is used to confirm gene deletions in Trp53, Pten, and Rb1. Immunofluorescence (IF) staining of organoids demonstrates positive expression of urothelial lineage markers (CK5 and p63). The organoids are injected subcutaneously into host mice for tumor expansion and serial passages. The immunohistochemistry (IHC) of xenografts exhibits positive expression of CK7, CK5, and p63 and negative expression of CK8 and Uroplakin 3. In summary, adenovirus-mediated gene deletion from mouse urothelial cells engineered with loxP sites is an efficient method to rapidly test the tumorigenic potential of defined genetic alterations.

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

This protocol describes the process of the generation and characterization of mouse urothelial organoids harboring deletions in genes of interest. The methods include harvesting mouse urothelial cells, ex vivo transduction with adenovirus driving Cre expression with a CMV promoter, and in vitro as well as in vivo characterization.

Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites ...

Engineering Oncogenic Heterozygous Gain-of-Function Mutations in Human Hematopoietic Stem and Progenitor Cells

Throughout their lifetime, hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations. Some of these mutations alter HSPC functional properties such as proliferation and differentiation, thereby promoting the development of hematologic malignancies. Efficient and precise genetic manipulation of HSPCs is required to model, characterize, and better understand the functional consequences of recurrent somatic mutations. Mutations can have a deleterious effect on a gene and result in loss-of-function (LOF) or, in stark contrast, may enhance function or even lead to novel characteristics of a particular gene, termed gain-of-function (GOF). In contrast to LOF mutations, GOF mutations almost exclusively occur in a heterozygous fashion. Current genome-editing protocols do not allow for the selective targeting of individual alleles, hampering the ability to model heterozygous GOF mutations. Here, we provide a detailed protocol on how to engineer heterozygous GOF hotspot mutations in human HSPCs by combining CRISPR/Cas9-mediated homology-directed repair and recombinant AAV6 technology for efficient DNA donor template transfer. Importantly, this strategy makes use of a dual fluorescent reporter system to allow for the tracking and purification of successfully heterozygously edited HSPCs. This strategy can be employed to precisely investigate how GOF mutations affect HSPC function and their progression toward hematological malignancies.

Novel strategies to faithfully model somatic mutations in hematopoietic stem and progenitor cells (HSPCs) are necessary to better study hematopoietic stem cell biology and hematological malignancies. Here, a protocol to model heterozygous gain-of-function mutations in HSPCs by combining the use of CRISPR/Cas9 and dual rAAV donor transduction is described.

Throughout their lifetime, hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations. Some of these mutations alter HSPC functional ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Immunology and Infection

Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting from advances in genome editing technology and lentivirus-mediated transgene delivery systems, an efficient and economical method is described here that establishes mice in which genes are manipulated specifically in hematopoietic stem cells. Lentiviruses are used to transduce Cas9-expressing lineage-negative bone marrow cells with a guide RNA (gRNA) targeting specific genes and a red fluorescence reporter gene (RFP), then these cells are transplanted into lethally-irradiated C57BL/6 mice. Mice transplanted with lentivirus expressing non-targeting gRNA are used as controls. Engraftment of transduced hematopoietic stem cells are evaluated by flow cytometric analysis of RFP-positive leukocytes of peripheral blood. Using this method, ~90% transduction of myeloid cells and ~70% of lymphoid cells at 4 weeks after transplantation can be achieved. Genomic DNA is isolated from RFP-positive blood cells, and portions of the targeted site DNA are amplified by PCR to validate the genome editing. This protocol provides a high-throughput evaluation of hematopoiesis-regulatory genes and can be extended to a variety of mouse disease models with hematopoietic cell involvement.

Described are protocols for the highly efficient genome editing of murine hematopoietic stem and progenitor cells (HSPC) by the CRISPR/Cas9 system to rapidly develop mouse model systems with hematopoietic system-specific gene modifications.

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting ...

Modeling Prostate Cancer in Genetically-engineered Mouse Models: A CRISPR/Cas9-mediated Localized Gene Editing Technique in Mouse Anterior Prostate Lobe Cells

Source: Riedel, M. et al. <a href="https://www.jove.com/video/57525" target="_blank">Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration.</a> J. Vis. Exp. (2018)
This video describes a method for editing specific genes in prostate gland cells using an adenovirus-based delivery system. The approach allows for orthotopic and localized alteration of gene expression utilizing CRISPR technology to develop novel mouse models for prostate cancer.

Source: Riedel, M. et al. Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration. J. Vis. Exp. (2018)
This video describes a method ...

JoVE Encyclopedia of Experiments

Prostate Cancer

Animal model studies

Isolation and Culture of Murine Tongue Epithelial Cells: A Procedure to Isolate and Culture Tongue Epithelial Cells from Mouse Model

Source: Prasad, M. et al. <a href="https://www.jove.com/video/60410" target="_blank">In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line Using an Adeno-Associated Virus-Cas9 System.</a> J. Vis. Exp. (2020)
In this video, we describe the isolation of epithelial cells from a murine tongue. These cells can be further cultured to give rise to cell colonies.

Source: Prasad, M. et al. In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line Using an Adeno-Associated Virus-Cas9 ...

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

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