基于CRISPR/Cas9系统构建Slfn2基因敲除的小鼠肺癌细胞系

doi:10.16088/j.issn.1001-6600.2021022302

Abstract

Abstract: To construct a stable knocking down of Slfn2 expression cell line in Lewis lung carcinoma (LLC) using CRISPR/Cas9 system, a guide RNA (sgRNA) sequence targeting the Slfn2 gene was designed by using the CHOPCHOP website software. After ligation of the sgRNA to the lenti-CRISPR-v2 plasmid, the T293 cells were co-transfected with pLenti CRISPR v2-Slfn2, pMD2.G and psPAX2 to generate Lentivirus. The packaged lentivirus was used to infect LLC cells, and Slfn2^-/-knockout LLC cells were obtained by screening and Monoclone culture. The results showed that the two SgRNAs were successfully inserted into the vector respectively. Slfn2 gene in LLC was successfully mutated by lentivirus packaged with three plasmids. Two Slfn2 knockout LLC cell lines were obtained, one with 8 bp deletion plus frameshift mutation and the other with 180 bp deletion mutation. The mouse lung cancer cells knocked out by Slfn2 gene were successfully constructed by CRISPR/Cas9 technology.

Key words: lung cancer, lentivirus, Slfn2, CRISPR/Cas9

CLC Number:

R734.2

CHEN Ying, ZHOU Zuping, XING Bing, PU Shiming. Construction of the Slfn2 Knockout Cell Lines in Lewis Lung Carcinoma Model Based on CRISPR/Cas9 System[J].Journal of Guangxi Normal University(Natural Science Edition), 2022, 40(4): 173-179.

References

[1] BADE B C, DELA CRUZ C S. Lung cancer 2020: epidemiology, etiology, and prevention[J]. Clinics in Chest Medicine, 2020, 41(1): 1-24. DOI: 10.1016/j.ccm.2019.10.001.
[2]MAO Y S, YANG D, HE J, et al. Epidemiology of lung cancer[J]. Surgical Oncology Clinics of North America, 2016, 25(3): 439-445. DOI: 10.1016/j.soc.2016.02.001.
[3]肖佳龙, 郑莹. 全球肺癌的流行及预防进展[J].中国癌症杂志, 2020, 30(10): 721-725. DOI: 10.19401/j.cnki.1007-3639.2020.10.001.
[4]WAN G Q, LIU Y H, ZHU J, et al. SLFN5 suppresses cancer cell migration and invasion by inhibiting MT1-MMP expression via AKT/GSK-3β/β-catenin pathway[J]. Cellular Signalling, 2019, 59: 1-12. DOI: 10.1016/j.cellsig.2019.03.004.
[5]KAGAMI T, YAMADE M, SUZUKI T, et al. The first evidence for SLFN11 expression as an independent prognostic factor for patients with esophageal cancer after chemoradiotherapy[J]. BMC Cancer, 2020, 20(1): 1123. DOI: 10.1186/s12885-020-07574-x.
[6]LIU F R, ZHOU P T, WANG Q, et. al. The Schlafen family: complex roles in different cell types and virus replication[J]. Cell Biology International, 2018, 42(1): 2-8. DOI: 10.1002/cbin.10778.
[7]KATSOULIDIS E, MAVROMMATIS E, WOODARD J, et al. Role of interferon α (IFN α-inducible Schlafen-5 in regulation of anchorage-independent growth and invasion of malignant melanoma cells[J]. The Journal of Biological Chemistry, 2010, 285(51): 40333-40341. DOI: 10.1074/jbc.M110.151076.
[8]KATSOULIDIS E, CARAYOL N, WOODARD J, et al. Role of Schlafen 2 (SLFN2) in the generation of interferon alpha-induced growth inhibitory responses[J]. The Journal of Biological Chemistry, 2009, 284(37): 25051-25064. DOI: 10.1074/jbc.M109.030445.
[9]MAVROMMATIS E, ARSLAN A D, SASSANO A, et al. Expression and regulatory effects of murine Schlafen (Slfn) genes in malignant melanoma and renal cell carcinoma[J]. The Journal of Biological Chemistry, 2013, 288(46): 33006-33015. DOI: 10.1074/jbc.M113.460741.
[10]OH P S, PATEL V B, SANDERS M A, et al. Schlafen-3 decreases cancer stem cell marker expression and autocrine/juxtacrine signaling in FOLFOX-resistant colon cancer cells[J]. American Journal of Physiology. Gastrointestinal and Liver Physiology, 2011, 301(2): G347-G355. DOI: 10.1152/ajpgi.00403.2010.
[11]HORTON M R, POWELL J D. Quieting T cells with Slfn2[J]. Nature Immunology, 2010, 11(4): 281-282. DOI: 10.1038/ni0410-281.
[12]MARZI L, SZABOVA L, GORDON M, et al. The indenoisoquinoline top1 inhibitors selectively target homologous recombination-deficient and Schlafen 11-positive cancer cells and synergize with olaparib[J]. Clinical Cancer Research, 2019, 25(20): 6206-6216. DOI: 10.1158/1078-0432.
[13]SCHWARZ D A, KATAYAMA C D, HEDRICK S M. Schlafen, a new family of growth regulatory genes that affect thymocyte development[J]. Immunity, 1998, 9(5): 657-668. DOI: 10.1016/s1074-7613(00)80663-9.
[14]刘梦伊, 周乔丹, 胡迎春, 等. Schlafen2基因在细胞中的表达及其生物学特性研究[J]. 军事医学科学院院刊, 2008, 32(3): 228-231. DOI: 10.3969/j.issn.1674-9960.2008.03.007.

Metrics

Viewed

Full text

Abstract

Cited

Shared

Discussed

Comments

Recommended 0

No Suggested Reading articles found!

Construction of the Slfn2 Knockout Cell Lines in Lewis Lung Carcinoma Model Based on CRISPR/Cas9 System

PDF (PC)

Abstract

Cite this article

share this article

References

Related Articles 3

Metrics

Comments

Recommended 0

[1]	ZHOU Jun, CHEN Shuman, XING Bing, CHEN Yajing, LI Yinling, HE Liu, ZHOU Zuping, PU Shiming. Antitumor Effect of Normal Mice Derived CD4⁺CD25⁺ Cells in Mice Lung Cancer Model [J]. Journal of Guangxi Normal University(Natural Science Edition), 2022, 40(2): 191-199.
[2]	LI Yinling, ZHOU Jing, CHEN Ying, CHEN Qiaoyuan, LIN Wanhua. Study on the Abnormal Expression of Sdr9c7 Gene in Erythrocytes of Tumor-bearing Mice [J]. Journal of Guangxi Normal University(Natural Science Edition), 2021, 39(6): 147-153.
[3]	MEI Han-bing, WANG Wei, YAO Xue, CHENG Lian-biao, HUANG Jin. Inhibitory Activity and Mechanism of Hsp90 Inhibitor Cynaroside Against Non-small Cell Lung Cancer in Vitro [J]. Journal of Guangxi Normal University(Natural Science Edition), 2015, 33(3): 91-97.