Optimization of transfection conditions for transfecting shRNA to endothelium of mouse angioma cells mediated by lentiviral vector / 中国综合临床

ABSTRACT

Objective To optimize the transfecfion parameters for transfecting small interfering RNA (siR-NA) to endothelium of mouse angioma (EOMA) cells mediated by lentiviral vector. Methods According to Poly-brene existing or not(8 μg/ml), series of multiplicity of infection (MOI) (1, 5, 10, 15, 20)and the time of infec-tion(4, 6, 12,24 h), EOMA cells were divided into 40 groups. 140 hours after transfection, cells were counted un-der fluorescent microscope to determine the percentage of the cells transfected under each condition. The cell viability was calculated by using Typan Blue. Results The percentage of positive cells was as high as 83% in the group with Polybrene (8 μg/ml), MOI(5) and the time of tranfectian (6 h), and the livability of EOMA in this group was 96%. After that, the transfection efficiency was not increased with the increasing of MOI, the time of transfection, but the mortality of EOMA was increased markedly. Conclusion The transfection efficiency and cell viability are dependent on the Polybrene(8 μg/ml),correct MOI (5)and the time of transfection (6 h).