Abstract: Objective: To investigate the long-term expression of the piggyback(PB) transposon system expressing human interleukin-6(IL-6), interleukin-3(IL-3), interleukin-15(IL-15), stem cell factor(SCF) and granulocyte-macrophage cstimulating Factor(GM-CSF) genes in the immunodeficient mice, and to provide a simple,long-term and new method for improving the reconstruction of human immune cells in the humanized mouse models. Methods: The PB transposon plasmid (PB-GFP) containing the GFP gene was constructed, and the PB transposon plasmid (PB-5F) containing human IL-6, IL-3, IL-15, SCF, and GM-CSF genes was constructed.The 293T cells were divided into negative control group(non-transfection), positive control group(transfected with pLVTHM), transient transfection group transfected with PB-GFP plasmid and stable transfection group[transfected with PB-GFP plasmid together with transposase plasmid (super-PB)].The proportions of GFP⁺ cells in varions groups were measured by flow cytometry every three days after transfection. The NOD.Cg-Prkdc^scid IL2rg^tm1Wjl/SzJ(NCG) mice were divided into transient transfection group and stable transfection group. The mice in transient transfection group were transfected with PB-GFP alone, and the mice in stable transfection group were transfected with PB-5F and super-PB. On the 1st, 4th, 5th and 9th days and the following every week after the transfection, the blood samples were collected, and the serum was separed;the levels of IL-6, IL-3, IL-15, SCF, and GM-CSF in serum of the mice in various groups were detected by ELISA. Results: At 30 d after transfection of PB-GFP, the percentage of GFP⁺ cells of the mice in stable transfection group (4.61%±0.42%) was significantly higher than those in positive control group (0.58%±0.05%) and transient transfection group (0.86%±0.10%) (P<0.05). At 30 d after transfection of PB-5F plasmid, the levels of serum IL-6, IL-15 and GM-CSF of the NCG mice in stable transfection group were significantly higher than those in transient transfection group (P<0.05); at 60 d after transfection, the levels of IL-6, IL-15 and GM-CSF in serum of the mice in stable transfection group were also found. Conclusion: The long-term expression of the PB (piggyBac) transposon system is verified in vitro, and the PB transposon system expressing human IL-6, IL-3, IL-15, SCF and GM-CSF genes is successfully constructed.An immunodeficient mouse model with long-term and stable expressions of human IL-6,IL-15,and GM-CSF is established.

Key words: humanized mouse of immune system, interleukin-6, interleukin-3, interleukin-15, stem cell factor, granulocyte-macrophage cstimulating factor, transposon