Construction of POR gene knockout,complementation and overexpression LO2 cell lines and preliminary application as AFB1 exposed model
To construct three LO2 cell lines separately with POR gene stable knockout(PORKO),complementation(PORCO),and overexpression(POROE),and provide cell models for further in-depth research on the function of the POR gene in the mechanism of AFB1 toxicity.Firstly,two sgRNAs targeting the POR gene were designed using CRISPR/Cas9 technology and cloned into the lentiCRISPR v2 vector,and then lentivirus packaging and virus infec-tion were performed.After screening LO2 PORKO monoclonal cells,they were sequenced and identified by Western blot.Secondly,the pcDNA3.1(+)/myc-His B-POR recombinant plasmid was constructed by homologous recombi-nation method,and the LO2 PORCO and LO2 POROE cell lines were respectively constructed through cell transfection and were identified by Western blot after screening with G-418.After the wild-type LO2,LO2 PORKO,LO2 PORCO,and LO2 POROE cell lines were exposed with 4 μmol·L-1 and 8 μmol·L-1 AFB1for 48 h,then the cell morphology was observed,and the cell viability was determined.The results showed that sgRNA-2 could successfully knock out the POR gene to obtain the LO2 PORKO cell line,and the LO2 PORCO and LO2 POROE cell lines were successfully constructed.After each cell line was exposed to 4 μmol·L-1 and 8 μmol·L-1 AFB1 for 48 h,compared with the wild-type LO2 cell viability rate of 43.66%±0.58%and27.90%±0.46%,the cell viability rate of LO2 PORKO cells was extremely significant,both 100%(P<0.000 1);the cell viability rate of LO2 PORCO cells was still signif-icantly different,respectively 57.07%±0.74%and 42.54%±0.68%(P<0.05);the cell viability rate of LO2 POROE cells was significantly lower,respectively 24.58%±0.92%and 17.99%±0.81%(P<0.01).In conclu-sion,this paper successfully constructed LO2 PORKO,LO2 PORCO,and LO2 POROE cell lines,and studied the cell viability rate and cell morphology changes of normal LO2,LO2 PORKO,LO2 PORCO,and LO2 POROE cell lines with AFB1exposure,and discovered that the POR gene has a huge impact on AFB1-induced cytotoxicity.This paper laid a foundation for the subsequent in-depth research on the function of the POR gene in the mechanism of AFB1 toxicity.
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