Establishment of auto-fluorescent K562 cell model using recombinant retrovirus vector

objective:To establish an auto-fluorescent K562 cell model using recombinant retroviral vector system. Methods:The full length cDNA of EGFP gene was inserted into the pCMV-hyg to construct recombinant retroviral plasmid pCMV-EGFP-hyg. The retrovirus containing EGFP was produced by co-transfection of T293 cell line with pCMV-EGFP-hyg, pCMV-G and pCMV-GP. The K562 cells expressing EGFP(EGFP-K562) were obtained by co-culture with recombinant retrovirus-producing T293 cells and Hyg selection. EGFP expression was examined with flow cytometry(FCM) and fluorescence microscopy. To show the application of EGFP-K562 in laboratory research, NK activity assay using EGFP-K562 as target cells was carried out in a series of effector(E) to target(T) cell ratio. Results:Recombined retrovirus containing EGFP was successfully constructed with high titers through co-transfection of T293 cells with pCMV-EGFP-hyg, pCMV-G and pCMV-GP. The highest titer of recombinant virus was 1.5×106CFU/mL when harvested at time point of 48 hours. Strong fluorescent in EGFP-K562 cells were developed and retained without any change during up to 20 passages of cell culture. More than 98% EGFP-K562 was achieved after selection with Hyg. NK activity assay employing EGFP-K562 as target cells showed peripheral blood mononuclear cells(PMNC) exhibited a different cytotoxicity with different E ∶ T ratio and incubation time. The optimal condition of NK activity assay was at a ratio of 40 ∶ 1 between E and T with 4 h incubation. Conclusion:Auto-fluorescent K562 cells was stably established which might provide a novel cell models in the experimental studies.