Construction of 4T1 CXCR4 knockout gene stable strain using CRISPR/Cas9 gene editing system

LIU Si-nian LUAN Jing-yang ZHANG Yan CAO Guan-hua CAO Guang-jin LI Ling*

doi:10.16098/j.issn.0529-1356.2018.03.006

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Acta Anatomica Sinica ›› 2018, Vol. 49 ›› Issue (3) : 303-308. DOI: 10.16098/j.issn.0529-1356.2018.03.006

Cell and Molecules Biology

Construction of 4T1 CXCR4 knockout gene stable strain using CRISPR/Cas9 gene editing system

LIU Si-nian¹LUAN Jing-yang¹ ZHANG Yan¹ CAO Guan-hua¹ CAO Guang-jin¹LI Ling ^1,2*

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Abstract

Objective To knock out CXCR4 gene in 4T1 cell by CRISPR/Cas9 system and to construct 4T1 cell strain with knocking out CXCR4 gene stably. Methods Two pairs of sgRNAs that could specifically identify the exons of CXCR4 gene were designed to construct LentiCRISPRv2-sgRNA recombinant plasmid and transformed into competent Stbl3.Then the recombinant plasmids were screened for sequencing and transfected into 293T cell to packaging into lentivirus. After infection of 4T1 cells with lentivirus, stable transfected cells were selected by puromycin,and monoclonal cells were isolated and cultured by limiting dilution method . The genomic DNA of monoclonal cells was extracted and sequenced. The expression of CXCR4 mRNA was detected by Real-time PCR. The expression of CXCR4 protein was detected by Western blotting. Results The LentiCRISPRv2-sgRNA recombinant plasmid was successfully constructed, and a cell strain of knocking out CXCR4 deletion 27 bp was obtained. The expression level of CXCR4 mRNA was reduced, and there was almost no expression of CXCR4 protein in cell strain. Conclusion Recombinant plasmids targeting the CXCR4 gene was obtained by CRISPR/Cas9 gene edit system, and the cell strain with stable knockout CXCR4 gene was successfully screened out.

Key words

CRISPR/Cas9 / Gene knockout / CXCR4 / 4T1 cell / Real-time PCR / Western blotting

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LIU Si-nian LUAN Jing-yang ZHANG Yan CAO Guan-hua CAO Guang-jin LI Ling. Construction of 4T1 CXCR4 knockout gene stable strain using CRISPR/Cas9 gene editing system[J]. Acta Anatomica Sinica. 2018, 49(3): 303-308 https://doi.org/10.16098/j.issn.0529-1356.2018.03.006

References

［1］Dewan MZ, Ahmed S, Iwasaki Y, et al. Stromal cell-derived factor-1 and CXCR4 receptor interaction in tumor growth and metastasis of breast cancer［J］. Biomed Pharmacother, 2006, 60(6):273-276.

［2］Müller A, Homey B, Soto H, et al. Involvement of chemokine receptors in breast cancer metastasis［J］. Nature, 2001, 410(6824):50-56.

［3］Ali S, Lazennec G. Chemokines: novel targets for breast cancer metastasis［J］. Cancer Metastasis Rev, 2007,26(34):401-420.

［4］Yoon Y, Liang Z, Zhang X, et al. CXC chemokine receptor-4 antagonist blocks both growth of primary tumor and metastasis of head and neck cancer in xenograft mouse models［J］. Cancer Res, 2007,67(15):7518-7524.

［5］Teicher B A, Fricker S P. CXCL12 (SDF-1)/CXCR4 Pathway in Cancer［J］. Clinical Cancer Res,2010,16(11):2927-2931.

［6］Wang YL,He XM, Tang W, et al. Effect of stromal cell derived factor-1α/CXCR4/CXCR7 axis on migration of the bone marrow mesenchymal stem cells［J］. Acta Anatomica Sinica, 2014,45(5):639-645.（in Chinese）

王玉兰，何晓梅，唐薇，等. 基质细胞衍生因子-1α/CXCR4/CXCR7轴对骨髓间充质干细胞迁移的影响［J］. 解剖学报,2014, 45(5):639-645.

［7] Liang Z, Brooks J, Willard M, et al. CXCR4/CXCL12 axis promotes VEGF-mediated tumor angiogenesis through Akt signaling pathway［J］. Biochem Biophys Res Commun, 2007, 3 (359):716-722.

［8］Wu J, Wu X, Liang W, et al. Clinicopathological and prognostic significance of chemokine receptor CXCR4 overexpression in patients with esophageal cancer: a meta-analysis［J］. Tumour Biol, 2014, 35(4):3709-3715.

［9］Han M, Lv S, Zhang Y, et al. The prognosis and clinicopathology of CXCR4 in gastric cancer patients: a meta-analysis［J］. Tumour Biol, 2014, 35(5):4589-4597.

［10］Lv S, Yang Y, Kwon S, et al. The association of CXCR4 expression with prognosis and clinicopathological indicators in colorectal carcinoma patients: a meta-analysis［J］. Histopathology,2014,64(5):701-712.

［11］Schimanski CC, Bahre R, Gockel I, et al. Dissemination of hepatocellular carcinoma is mediated via chemokine receptor CXCR4［J］. Br J Cancer, 2006,95(2):210-217.

［12］Billadeau DD, Chatterjee S, Bramati P, et al. Characterization of the CXCR4 signaling in pancreatic cancer cells［J］. Int J Gastrointest Cancer, 2006,37(4):110-119.

［13］Miki J, Furusato B, Li H, et al. Identification of putative stem cell markers, CD133 and CXCR4, in hTERT-immortalized primary nonmalignant and malignant tumor-derived human prostate epithelial cell lines and in prostate cancer specimens［J］. Cancer Res, 2007,67(7):3153-3161.

［14］Xu C, Zhao H, Chen H, et al. CXCR4 in breast cancer: oncogenic role and therapeutic targeting［J］. Drug Des Devel Ther, 2015, 9(1):4953-4964.

［15］Liang Z, Bian X, Shim H. Inhibition of breast cancer metastasis with microRNA-302a by downregulation of CXCR4 expression［J］. Breast Cancer Research and Treatment, 2014, 146(3):535-542.

［16］Liu R, Tan YZh, Wang HJ, et al. The effects of cytokines on the migration and mobilization of lympahatic endothelial progenitor cells［J］. Acta Anatomica Sinica, 2008, 39(1):45-49.(in Chinese)

刘锐，谭玉珍，王海杰，等. 细胞因子对淋巴管内皮祖细胞的趋化和动员作用［J］. 解剖学报,2008,39(1):45-49.

［17］Chen DP, Zhang ZhJ, Wu XL, et al. Construction of rat CXCR4 gene RNAi lentivirus vector and its expression in bone marrow mesenchymal stem cells［J］. Chinese Journal of Biotechnology, 2009, 25(2):299-305.(in Chinese)

陈东平，张志坚，吴秀丽，等. 大鼠CXCR4基因RNAi慢病毒载体的构建及其在骨髓间质干细胞中的表达［J］. 生物工程学报,2009,25(2):299-305.

［18］Liang Z, Yoon Y, Votaw J, et al. Silencing of CXCR4 blocks breast cancer metastasis［J］. Cancer Res,2005,65(3):967-971.

［19］Jiang K, Li J, Yin J, et al. Targeted delivery of CXCR4-siRNA by scFv for HER2(+) breast cancer therapy［J］. Biomaterials, 2015, 59(1):77-87.

［20］Sasaki H, Yoshida K, Hozumi A, et al. CRISPR/Cas9-mediated gene knockout in the ascidian Ciona intestinalis ［J］. Dev Growth Differ, 2014, 56(7):499-510.

［21］Fu Y, Foden JA, Khayter C, et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells ［J］. Nat Biotechnol, 2013, 31(9):822-826..

［22］Li K, Wang G, Andersen T, et al. Optimization of genome engineering approaches with the CRISPR/Cas9 system［J］. PLoS One, 2014, 9(8):e105779.

［23］Rath D, Amlinger L, Rath A, et al. The CRISPR-Cas immune system: biology, mechanisms and applications［J］. Biochimie, 2015, 117(1):119-128.

［24］Trevino AE, Zhang F. Genome editing using Cas6 nickases ［J］. Methods Enzymol, 2014, 546(1):161-174.

［25］Jinek M, Chylinski K, Fonfara I, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity［J］. Science, 2012, 337(6096):816-821.