Construction of FOXA2 Expression Tracer System Based on CRISPR/Cas9 System

doi:10.13560/j.cnki.biotech.bull.1985.2018-0111

Abstract

Abstract: This study enabled the tracing of FOXA2 gene on and off by using CRISPR/Cas9 system to insert a green fluorescent protein（GFP）nucleic acid sequence following the FOXA2 protein coding region. The study was carried out from the following five aspects：targeting sequence selection,cloning of Cas9-targeting plasmids and donor fragment,cells transfection and flow cytometry fluorescence sorting,limited dilution of positive cells,as well as enrichment culture and identification,transcription and translation level verification of monoclonal cells. The results showed that the expression of GFP was positive in the edited breast cancer cell line MCF-7. The expression level of GFP in breast cancer cells was positively correlated with the expression level of FOXA2 protein,and the expression of GFP and FOXA2 decreased synchronously after the induction by EGF,an inducing factor in the process of tumor cells epithelial-mesenchymal transition（EMT）. The results indicate that CRISPR/Cas9 targeting FOXA2 and using intracellular homology directed repair（HDR）to insert GFP sequence are success. Using the expression level of reporter gene protein for tracing the target gene on and off as well as its expression is significantly accurate and convenient.

Key words: CRISPR/Cas9, gene editing, FOXA2, tracing, breast cancer, MCF-7, epithelial-mesenchymal transition

CHENG Hao-jie, ZHANG Zhen-wang, TAN Gui-xiang, LIU Yu-xiang, PIAO Hui-tong, TAN Yong-jun. Construction of FOXA2 Expression Tracer System Based on CRISPR/Cas9 System[J]. Biotechnology Bulletin, 2018, 34(5): 80-86.

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