中国生物工程杂志

Objectives: To compare the efficiency of HL60 cellular transfection by eukaryotic expressing vector using different methods, and then the most suitable way for transfection could be screened out. Methods: Transfected the empty eukaryotic expressing vector into HL60 cells using 2mm and 4mm electroporating cups respectively using different instrumental parameter, following which the parameter could be ascertained based on the survival rate of HL60 cells. The comparison of transfection efficiency after 48h cellular culture was performed between groups with different plasmid addition and DMSO(dimethyl sulfoxide) supplement. The G418 was employed to screen out positive clones, and its concentration was chosen based on positive rate analysis. At last, pDsRED-C1 empty expressing vector was transfected into HL60 cells following the methods above, and cellular biocharacters could be observed using flow cytometry(FCM), cellular chemical staining and transmission electron microscope. What’s more, the same transfecting procedure was used to transfected another empty eukaryotic expressing vector eYFP into HL60 cells. Results: The death rate of HL60 cells was increased in line with the elevation of intensity of electric shock and frequency of pulse. Square wave was stronger in electroporation than convolution wave. Along with the addition of plasmid, the number of positive transfected HL60 cells was up-regulated at first, and downregulated subsequently with the constant tansfected parameter. Moreover, the positive transfected HL60 cells was much more in 2mm electroporated cup than 4mm electroporated cup under the same plasmid addition. The supplyment of DMSO in refrigerated transfection system could significantly improve the positive rate of transfection about nearly 13 times. 400μg/ml G418 was choosen as the best screening drug concentration. After cellular electroporation, the cellular surface antigen CD11b and CD14 could not be detected, and that, the apoptosis could not be found by observation of cellular morphology and ultramicrostructure. Same procedure was successfully used in the transfection of eYFP-C1 into HL60 cells. Conclusion: The modified electroporating procedure could significantly promote the positive rate of cellular transfection, which would lay the foundation of researchers’ following experiments.