Construction of immunodeficient mouse models with long-term expression of human cytokines based on piggyBac transposon system / 吉林大学学报(医学版)

ABSTRACT

Objetive To investigate the long-term expression of the piggyback (PB) transposon system expressing human interleukin-6 (IL-6), nterleukin-3 (IL-3), interleukin-15 (IL-15), stem cell factor (SCF) and granulocyte-macrophage ctimulang 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) contaning 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), postive 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-Prkdcscid IL2rgun1wj1/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 followng 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 GFP1 cells of the mice in stable transfection group (4. 61% + 0.42%) was significantly higher than those in postive 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 (Ptransfection, 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.