Strategies For Long Term Expression Of Multiple Human Cytokines In Immunodeficient Mice Based On Hydrodynamic Injection Of Transposon Vectors

Objective:The immune system humanized mice is one of the most widely used humanized mice models.It has been recognized as the effective small animal model for research in the field of biomedicine.After decades of research and development,the model has gradually developed.However,some cytokines were found to exhibit significant sequence divergence between human and mouse and to lack functional crossreactivity in vitro experiment.This leads to the lack of critical human species-specific signals that support human cell survival,development and function in humanized mice.It has been reported that IL-4,GM-CSF,CSF,IL 3 and other cytokines are species-specific and show no effect on human cells.The addition of exogenous IL 15,EPO and IL 3 in humanized mice could improve the reconstitution of human NK cells and red blood cells.Therefore,the supplementation of appropriate cytokines could be a way to increase the reconstitution of the myeloid and erythroid cells in humanized mice models by providing relative signals.This study was designed to optimize the human immune system in humanized mice by hydrodynamic injection of plasmid encoding and expressing multiple human cytokines that are IL 6,IL 3,IL 15,SCF,GM-CSF.Methods:An expression vector that contains multiple human cytokine genes,including IL-6,IL-3,IL-15,SCF,GM-CSF,was synthesized by molecular biology methods;then we confirmed the synthesis effect of the plasmid by agarose gel electrophoresis and gene sequencing: the single clone with target gene was screened by agarose gel and then through Nhel & BamHI double enzyme digestion.The sequnence of target clone was sequencing by COMATE company.The plasmid DNA with green fluorescent protein of transponson PiggyBac(PB-GFP)and the Super PB were co-transformed into 293 T cells.Then,we determined the expression of green fluorescent protein by fluorescence microscopy and flow cytometric analysis;plasmid expressing multiple human cytokine genes(50 μg/mouse)and Super PB(20 μg/mouse)were then mixed together and injected into immunodeficient mice(NCG)hydrodynamically.The expression levels of human IL-6,IL-3,IL-15,SCF and GM-CSF in mice serum were test by enzyme linked immunosorbent assay(ELISA)at the indicated time points.Results:The results of double digestion and agarose gel electrophoresis have suggested that PB-5F plasmid has correct size and sequences.Cell transfection experiment showed that there was no significant difference of GFP expression between the transient transfection group and the stable transfection group within 7 days after transfection;subsequently,the GFP expression was hardly detected in the transient transfection group,while the GFP of the stable transfection group could be continued expression for more than 30 days.Human cytokines in serum of mice showed no statistically different between the transient transfection group and the stable transfection group at the 5th day after injection,which were consistent with the cell transfection result;after 2 months,the expression of human cytokines could still be test in the stable transfection group.Conclusions: This study proved that:(1)The PB transposon plasmid(PB-5F)containing IL 6,IL 3,IL 15,SCF,and GM-CSF genes was successfully constructed;(2)PiggyBac(PB)transponson and transposase co-transformation system can maintain the long-term expression of human cytokines in vivo and in vitro.