基于CRISPR/Cas9技术的法尼醇X受体缺失株的构建及其对HeLa细胞增殖的影响

doi:10.16431/j.cnki.1671-7236.2021.02.006

Abstract

Abstract: This study was aimed to construct a stable FXR gene knockout cell line and its effect on HeLa cell proliferation by using CRISPR/Cas9 gene editing technique.According to the design rules of CRISPR/Cas9 target,three pairs of upstream and downstream small guide RNA (sgRNA) specifically identifying the first exon related sequence of FXR gene were designed,and the recombinant eukaryotic expression plasmid was constructed using PX459 plasmid as the vector.After enzymatic digestion and sequencing identification,the recombinant plasmid was transfected into HeLa cells,puromycin was used for positive cell screening,then the expression level of FXR in the stable FXR knockout strain of HeLa cells was detected by Real-time PCR,and the FXR knockout effect of cells was identified by Western blotting.Finally,the effect of FXR gene knockout on HeLa cell proliferation was detected by crystal violet staining.The results showed that after sequencing,three pairs of sgRNA were inserted in the correct position and direction PX459 plasmid vector,and successfully constructed the recombinant expression vector PX459-sgRNA.The results of Real-time PCR and Western blotting showed that FXR protein was not expressed in the selected cells after transfection,indicating that a stable FXR knockout cell line was constructed.Crystal violet results found FXR knockout HeLa dyeing depth was significantly lower than the normal HeLa.The D_{595 nm} value of FXR knockout HeLa cell hole was extremely significantly lower than that of wild-type HeLa cell hole (P<0.01),indicating that the proliferation capacity of FXR knockout HeLa cell strain was extremely significantly lower than that of normal HeLa cells.Endogenous FXR gene knockout cell lines were successfully obtained by CRISPR/Cas9 technology,and FXR gene had inhibitory effect on the proliferation of cancer cells.This experiment provided an effective tool for studying the function and mechanism of FXR and laid a foundation for studying the occurrence and development of FXR in related cancers.

Key words: CRISPR/Cas9; farnesiod receptor (FXR); HeLa cell; cell proliferation

CLC Number:

MA Fangxue, AN Yanan, WANG Chao, LI Yuan, LUAN Wenjing, WANG Xuefei, NI Lihui, ZHOU Hong, LIANG Junchao, YU Lu. Construction of FXR Deletion Strain Based on CRISPR/Cas9 Technology and Its Effect on HeLa Cell Proliferation[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(2): 450-456.

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Construction of FXR Deletion Strain Based on CRISPR/Cas9 Technology and Its Effect on HeLa Cell Proliferation

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