稳定过表达XAF1基因鼻咽癌放射抗拒CNE-2R细胞株的构建

Optimizing construction of a nasopharyngeal cancer CNE-2R cell stably expressing XAF1

Objective To examine methods for constructing a nasopharyngeal cancer cell line( CNE-2R) stably expressing( X-linked inhibitor of apoptosis associated factor 1) XAF1. Method CNE-2R cells were infected with lentivirus encoding XAF1 as well as a green fluorescent protein reporter. Cells were infected at multiplicities of infection ( MOI) of 50 or 100 without polybrene, or at an MOI of 100 in the presence of polybrene( 5 μg/mL) . Expression of XAF1 mRNA and protein was verified using quantitative RT-PCR and Western blotting. Results Faint fluorescence was detected in 60%of CNE-2R cells after transfection with lentivirus at an MOI of 50. A slightly higher percentage( 70%) of CNE-2R cells showed weak fluorescence after transfection with lentivirus at an MOI of 100. When cells were infected at an MOI of 100 in the presence of polybrene, 90% of CNE-2R cells showed strong fluorescence. Stably transfected cell lines were selected using puromycin. Conclusion The efficiency with which XAF1-expressing lentivirus can infect CNE-2R cells is closely related to the state of the cells, MOI, and polybrene concentration. Addition of appropriate concentrations of polybrene to lentivirus can increase efficiency of infection. Stably transfected cell lines can be selected with puromycin.