使用腺相关病毒-Cas9系统在Vitro建立基因工程的毛因头和颈部癌细胞系
1The Shraga Segal Department of Microbiology, Immunology and Genetics,Ben-Gurion University of the Negev, 2Faculty of Health Sciences,Ben-Gurion University of the Negev, 3Human Oncology & Pathogenesis Program (HOPP),Memorial Sloan Kettering Cancer Center, 4Institute of Pathology,Barzilai University Medical Center
Summary
为了了解肿瘤，需要开发在头颈部癌症患者中发生特定基因变异的鼠模型。在这里，我们提出了一个方案，用于使用腺相关病毒-Cas9系统对原性鼠舌细胞进行体外转化，以产生具有特定基因组改变的小鼠HNC细胞系。
Abstract
使用原发正常上皮细胞，利用聚集调控间隔，在肿瘤基因和肿瘤抑制基因中引入特定突变，可以重新诱导细胞转化所需的基因组改变基于小鼠的短音重复（CRISPR）基因组编辑技术。这项技术使我们能够准确地模拟使用小鼠发生的人类癌症的基因变化。通过基因转化鼠原细胞，我们可以更好地研究癌症的发展、进展、治疗和诊断。在这项研究中，我们使用可诱导的Cas9小鼠舌上皮细胞，在体外使用腺相关病毒（AAV）进行基因组编辑。具体来说，通过改变正常舌上皮细胞中的KRAS、p53和APC，在体外产生了一个小鼠头颈部癌（HNC）细胞系，这是合成小鼠的肿瘤。这里介绍的方法详细介绍了如何生成具有特定基因组改变的HNC细胞系，并解释了它们是否适合预测合成小鼠的肿瘤进展。我们设想，这种有前途的方法将是翔实和有用的研究肿瘤生物学和HNC的治疗。
Introduction
HNC是一种常见的恶性肿瘤，全世界1.模拟HNC新奇的起源目前处于一个科学的转折点2。虽然在HNC2、3、4（例如TP53、PIK3CA、NOTCH1、FAT1和RAS）中发现了许多基因突变，但诱导HNC所需的基因组突变的具体组合仍不清楚。
目前人类HNC细胞系的使用大大有助于阐明与发病机制和治疗相关的机制3。然而，研究免疫功能低下的鼠系中的人类细胞系有其局限性，因为这些系统没有解决体内肿瘤过程、特定基因突变的作用以及免疫微环境中的治疗反应。因此，具有特定基因改变的鼠细胞系的发育和建立对于研究不同基因如何促进转化过程以及测试免疫能力小鼠的新型分子疗法至关重要。
生物医学研究中的基因功能研究受到DNA编辑技术的进步的显著影响，例如引入双链断裂（DSBs）。这些技术，包括使用锌指核酸酶、转录活化剂样效应核酸酶和聚类调节间隔短回触性重复（CRISPR/Cas9），允许在其位点操作任何感兴趣的基因。最新的CRISPR/Cas9系统由引导RNA（gRNA）组成，该RNA指示Cas9核酸酶在基因组中的特定位点生成DSB。该系统在修改任何细胞或目标组织的内源性基因方面得到了广泛的认可，即使在最传统的难以治疗的有机体5中也是如此。由于其简单性、速度和效率，它比其他系统具有多种优势。
在肿瘤学领域，CRISPR/CAS9技术满足了有效模仿癌细胞的需求。为了在HNC建立这个系统，我们操纵了有效的KRAS肿瘤基因和两个重要的肿瘤抑制基因，APC和p536。根据GENIE数据库7，这种组合在HNC是罕见的。RAS 突变（HRAS、NRAS 和 KRAS）仅在所有 HNC 人群中存在 +7%。这些肿瘤往往对治疗8，9具有抗药性。
Cas9及其gRNA的传递是通过使用AAV或慢病毒的病毒转导实现的。重组AAV通常是向目标细胞传递基因的首选方法，因为它的度子含量高，免疫反应温和，能够换出广泛的细胞范围，而且整体安全。使用AAV系统，已经生成了各种组织特异性小鼠细胞系，新的细胞系仍在开发中。然而，一个高效的基因组编辑系统，可以产生鼠HNC细胞系模型细胞仍有待开发。在这项研究中，我们寻求开发一种基于体外AAV-Cas9的系统，用于将原生鼠舌细胞转化为肿瘤状态。这种独特的CRISPR/Cas9转化方案和已建立的肿瘤细胞系可用于更好地了解由基因组变化引起的HNC生物学。
Protocol
这项研究得到了内盖夫动物护理和使用委员会的本古里安大学的批准。动物实验获得IACUC（IL.80-12-2015和IL.29-05-2018（E））的批准。本研究中使用的动物试验、住房和环境条件的所有方面都符合《实验室动物护理和使用指南》13。 1. 阿德诺相关病毒生产 第1天：细胞培养 种子 4 x 106 HEK293T 细胞每 14.5 厘米板在 15 mL 的 DMEM.准备10个板，用于用…
Representative Results
使用 AAV 系统转换正常的 Cas9 单元图1提供了本研究中使用的AAV转基因质粒的详细矢量图。图 2概述了基于 AAV-Cas9 的系统的设计与工作。为了产生病毒颗粒，使用PEI转染方法用AAV转录载体和其他病毒包装载体转染HEK293T细胞。转染后，对含病毒细胞进行收集和分离，采用肝素-腺素纯化工艺，对病毒颗粒进行纯化和?…
Discussion
以前已经使用几种方法来转化培养中具有可变成功率的初级细胞25，26，27，28。这些方法大多使用小鼠成纤维细胞进行转化14、17、18、19或使用致癌物质，如4-硝基基苯丙胺-1-氧化物（4-NQO）21，22用于开发鼠细胞系模型。<sup…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们要感谢丹尼尔·吉特勒博士为我们提供了pAd Delta 5帮手质粒。这项工作由以色列科学基金会（ISF，700/16）（致ME）、美国-以色列两国科学基金会（BSF，2017323）（对ME和MS）、以色列癌症协会（ICA，20170024）（对ME）、以色列癌症研究基金会（ICRF，17-1693-RCDA）（对ME）和关注基金会（#7895）资助。研究金：阿隆奖学金，ME和BGU克雷特曼奖学金给SJ和MP。
Materials
| Antibodies | |||
| Anti mouse HRP | Jackson ImmunoResearch | 115-035-146 | |
| Anti rabbit HRP | Jackson ImmunoResearch | 711-035-152 | |
| Cas9 Mouse mAb | Cell Signaling Technology | 14697 | |
| Cre | BioLegend | 900901 | |
| Cy3-AffiniPure Goat Anti-Mouse IgG | Jackson ImmunoResearch | 115-165-062 | |
| Cy-AffiniPure Goat Anti-Rabbit IgG | Jackson ImmunoResearch | 111-165-144 | |
| GFP | Santa Cruz Biotechnology | sc-9996 | |
| Phospho-p44/42 MAPK (Erk1/2) | Cell Signaling Technology | 4370 | |
| Rabbit monoclonal anti E cadherin | Cell Signaling Technology | 3195S | |
| Rabbit monoclonal anti-KRT 14 | Abcam | AB-ab181595 | |
| β actin | MP Biomedicals | 691001 | |
| β catenin | Cell Signaling Technology | 9582S | |
| Cell lines | |||
| HEK93T | ATCC | CRL-3216 | |
| Culture Media, Chemicals and Reagents | |||
| Bradford Reagent | Bio-Rad | 30015484 | |
| BSA | Amresco | 0332-TAM-50G | |
| DAPI fluoromount | Southern Biotech | 0100-20 | |
| DMEM | Biological Industries Israel Beit-Haemek Ltd. | 01-055-1A | |
| ECL (Westar Supernova and Westar Nova 2.0) | Cyanagen | XLS3.0100 and XLS071.0250 | |
| FBS | Biological Industries Israel Beit-Haemek Ltd. | 04-127-1A | |
| HBSS | Sigma | H6648 | |
| Heparin – Agarose | Sigma | H6508 | |
| Isolate II Genomic DNA Kit | Bioline | BIO-52066 | |
| MgCl2 | Panreac AppliChem | 300283 | |
| NaCl | Bio Lab Ltd | 1903059100 | |
| PBS | Biological Industries Israel Beit-Haemek Ltd. | 02-023-1A | |
| PEI | Polysciences | 23966-1 | |
| Pen Strep Solution | Biological Industries Israel Beit-Haemek Ltd. | 03-031-1B | |
| PFA | Santa Cruz Biotechnology | 30525-89-4 | |
| Phosphatase inhibitor cocktail | Biotool | B15001A/B | |
| Protease inhibitor cocktail | MilliporeSigma | P2714-1BTL | |
| Tris buffer | MERCK Millipore | 648311-1KG | |
| Enzymes | |||
| Benzonase | Sigma | E1014 | |
| Collagenase IV | Thermo Fisher Scientific | 17104019 | |
| DNAse | Thermo Fisher Scientific | 18047019 | |
| Hyaluronidase | MilliporeSigma | H3506 | |
| Trypsin | Biological Industries Israel Beit-Haemek Ltd. | 03-050-1B | |
| Glass wares | |||
| Cover slips | Bar Naor | BNCB00130RA1 | |
| Slides | Bar Naor | BN9308C | |
| Mouse strains | |||
| C57BL/6 | Envigo | ||
| B6;129-Gt(ROSA)26Sortm1(CAG-cas9*,-EGFP)Fezh/J | Jackson labs | 24857 | |
| NOD.CB17-Prkdc-scid/NCr Hsd (Nod.Scid) | Envigo | ||
| Plasmids | |||
| AAV pCM109 EFS Cre sg APC sg Kras sg P53- Kras HDR | Broad Institute of MIT | Kind gift from Dr Randall J Platt and Dr. Joseph Rosenbluh, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA | |
| AAV 2/9 capsid vector | Addgene | 112865 | |
| pAD Delta F5 helper | Ben Gurion University of the Negev | 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. | |
| Plastic wares | |||
| Amicon-ULTRA filter 100 KDa | Millipore | UFC910024 | |
| 0.22 µm sterile filters, 4 mm | Millex | SLGV004SL | |
| 0.45 µm sterile filters, 13 mm | Millex | SLHV013SL | |
| Culture plates | Greiner Bio-One | ||
| Falcon tubes | Greiner Bio-One |
References
- Reyes-Gibby, C. C., et al. Genome-wide association study identifies genes associated with neuropathy in patients with head and neck cancer. Scientific Reports. 8 (1), 8789 (2018).
- Riaz, N., Morris, L. G., Lee, W., Chan, T. A. Unraveling the molecular genetics of head and neck cancer through genome-wide approaches. Genes & Diseases. 1 (1), 75 (2014).
- Leemans, C. R., Snijders, P. J. F., Brakenhoff, R. H. The molecular landscape of head and neck cancer. Nature Reviews Cancer. 18 (5), 269-282 (2018).
- Jiang, X., Ye, J., Dong, Z., Hu, S., Xiao, M. Novel genetic alterations and their impact on target therapy response in head and neck squamous cell carcinoma. Cancer Management and Research. 11, 1321-1336 (2019).
- Im, W., Moon, J., Kim, M. Applications of CRISPR/Cas9 for Gene Editing in Hereditary Movement Disorders. Journal of Movement Disorders. 9 (3), 136-143 (2016).
- Li, H., et al. Genomic analysis of head and neck squamous cell carcinoma cell lines and human tumors: a rational approach to preclinical model selection. Molecular Cancer Research: MCR. 12 (4), 571-582 (2014).
- AACR Project GENIE Consortium, T.A.P.G.. AACR Project GENIE: Powering Precision Medicine through an International Consortium. Cancer Discovery. 7 (8), 818-831 (2017).
- Suh, Y., Amelio, I., Guerrero Urbano, T., Tavassoli, M. Clinical update on cancer: molecular oncology of head and neck cancer. Cell Death & Disease. 5 (1), e1018 (2014).
- Anderson, J. A., Irish, J. C., Ngan, B. Y. Prevalence of RAS oncogene mutation in head and neck carcinomas. The Journal of Otolaryngology. 21 (5), 321-326 (1992).
- Ryals, R. C., Boye, S. L., Dinculescu, A., Hauswirth, W. W., Boye, S. E. Quantifying transduction efficiencies of unmodified and tyrosine capsid mutant AAV vectors in vitro using two ocular cell lines. Molecular Vision. 17, 1090-1102 (2011).
- Smith-Arica, J. R., et al. Infection Efficiency of Human and Mouse Embryonic Stem Cells Using Adenoviral and Adeno-Associated Viral Vectors. Cloning and Stem Cells. 5 (1), 51-62 (2003).
- Li, J., Xiao, X., Kenniston, T., Kudlow, J., Giannoukakis, N. AAV Vectors. Molecular Therapy. 3 (5), S174-S192 (2001).
- Institute of Laboratory Animal Resources (U.S.). . Guide for the care and use of laboratory animals. , (1996).
- Platt, R. J., et al. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell. 159 (2), 440-455 (2014).
- Zhang, Y., Chirmule, N., Gao, G. P., Wilson, J. CD40 ligand-dependent activation of cytotoxic T lymphocytes by adeno-associated virus vectors in vivo: role of immature dendritic cells. Journal of Virology. 74 (17), 8003-8010 (2000).
- Xiao, X., Li, J., Samulski, R. J. Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus. Journal of Virology. 72 (3), 2224-2232 (1998).
- Auricchio, A., Hildinger, M., O’Connor, E., Gao, G. P., Wilson, J. M. Isolation of Highly Infectious and Pure Adeno-Associated Virus Type 2 Vectors with a Single-Step Gravity-Flow Column. Human Gene Therapy. 12 (1), 71-76 (2001).
- Lock, M., et al. Characterization of a Recombinant Adeno-Associated Virus Type 2 Reference Standard Material. Human Gene Therapy. 21 (10), 1273 (2010).
- Challis, R. C., et al. Systemic AAV vectors for widespread and targeted gene delivery in rodents. Nature Protocols. 14 (2), 379-414 (2019).
- Rabinowitz, J. E., et al. Cross-packaging of a single adeno-associated virus (AAV) type 2 vector genome into multiple AAV serotypes enables transduction with broad specificity. Journal of Virology. 76 (2), 791-801 (2002).
- Cheng, D. T., et al. Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT). The Journal of Molecular Diagnostics. 17 (3), 251-264 (2015).
- Sun, H., Taneja, R. Analysis of Transformation and Tumorigenicity Using Mouse Embryonic Fibroblast Cells. Cancer Genomics and Proteomics. 383, 303-310 (2007).
- Durkin, M., Qian, X., Popescu, N., Lowy, D. Isolation of Mouse Embryo Fibroblasts. BIO-PROTOCOL. 3 (18), (2013).
- Jozefczuk, J., Drews, K., Adjaye, J. Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells. Journal of Visualized Experiments. (64), e3854 (2012).
- Boehm, J. S., Hession, M. T., Bulmer, S. E., Hahn, W. C. Transformation of Human and Murine Fibroblasts without Viral Oncoproteins. Molecular and Cellular Biology. 25 (15), 6464 (2005).
- Gupta, T., Sáenz Robles, T. M., Pipas, J. M. Cellular transformation of mouse embryo fibroblasts in the absence of activator E2Fs. Journal of Virology. 89 (9), 5124-5133 (2015).
- Leong, H., Blewitt, M. Retrovirus Mediated Malignant Transformation of Mouse Embryonic Fibroblasts. BIO-PROTOCOL. 3 (15), (2013).
- Parikh, N., Nagarajan, P., Sei-ichi, M., Sinha, S., Garrett-Sinha, L. A. Isolation and characterization of an immortalized oral keratinocyte cell line of mouse origin. Archives of Oral Biology. 53 (11), 1091-1100 (2008).
- Badarni, M., et al. Repression of AXL expression by AP-1/JNK blockage overcomes resistance to PI3Ka therapy. JCI Insight. 5, 125341 (2019).
- Chen, Y. F., et al. Establishing of mouse oral carcinoma cell lines derived from transgenic mice and their use as syngeneic tumorigenesis models. BMC Cancer. 19 (1), 281 (2019).
- Hoover, A. C., et al. The Role of Human Papillomavirus 16 E6 in Anchorage-Independent and Invasive Growth of Mouse Tonsil Epithelium. Archives of Otolaryngology-Head & Neck Surgery. 133 (5), 495 (2007).
- Smahel, M., et al. Metastatic MHC class I-negative mouse cells derived by transformation with human papillomavirus type 16. British Journal of Cancer. 84 (3), 374-380 (2001).
- He, L., et al. Increased Growth of a Newly Established Mouse Epithelial Cell Line Transformed with HPV-16 E7 in Diabetic Mice. PloS One. 11 (10), e0164490 (2016).
Cite This Article
Prasad, M., Jagadeeshan, S., Scaltriti, M., Allon, I., Elkabets, M. In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System. J. Vis. Exp. (155), e60410, doi:10.3791/60410 (2020).