Establishment Of Induced Pluripotent Stem Cell-like Cells From Guinea Pig

Induced pluripotent stem cells(iPSCs) is refered to the reprogramming of somatic cells to a pluripotent state whereby they adopt characteristics and function of embryonic stem cells. Induced pluripotent stem cells can renew itself and differentiate into various types of cells. It not only have important value in the replacement therapy, but also can be used for the establishment of disease model in vitro. It is convenient for mechanisms of disease research, drug monitoring as well as the inspection of new treatments. Because guinea pigs have some special anatomical physiology characteristics and biological features, so they are widely used in biomedical fields. So guinea pig can serve as ideal animal model of pathogenesis in disease research and drug screening. But so far, there has been no reports of guinea pig iPS cells research. In this research, we used the reprogramming method of four transcription factors (oct4, sox2, klf4 & c-myc) on fibroblasts from embryo guinea pig with the lentivirus as carrier and get guinea pig iPS cell lines. It lay a foundation for further establishment of animal disease model and the drug screening. The experimental results are as follows:1.Mouse embryonic fibroblast and guinea pig fetal fibroblasts are isolated respectively by organizing block adherent method, the cells passage culture are stable. After the virus infection and multiple passage culture of guinea pigs embryo fibroblasts, cell cycle is still normal.’So it can be the reprogrammed target cells.2.Transfection of three plasmids into 293T cells, using four different transfection reagents:calcium phosphate, liposome 2000, liposome LTX and QuickShuttle-293 reagent, then we compared the packing efficiency and toxic effects of recombinantional virus vector, which encode green fluorescent protein (GFP) gene. And we also compared the infection ability of packaging virus with guinea pig fetal fibroblasts. Positive cells and cell activity number are tested respectively by flow cytometry technique and MTT method, and finally reveals the QuickShuttle-293 has a good effect in transfection efficiency during the process of packaging, cytotoxicity and virus infection efficiency after packaging.3.Traditional reprogramming method of four transcription factors (oct4, sox2, klf4 & c-myc) is used on guinea pig embryo fibroblasts with the recombinational lentivirus as carrier and get guinea pig iPS cell lines. Comparison of the three kinds of small molecules (vitamin C, valproic acid sodium, butyrate sodium) is made for the promotion of cell reprogramming, eventually we found that adding vitamin C and sodium valproic acid medium can improved the efficiency on the reprogramming significantly. And we have the biological characteristics detection on guinea pig iPS clones:(1)Alkaline phosphatase staining of guinea pig iPS sample cells, the result was positive.(2)RNA is extracted of guinea pig iPS cell, RT-PCR detection of marker genes related to ES cells, the result shows that the iPS has expression of endogenous gene:oct4, sox2, klf4, c-myc and nanog, and the expression of genes are much higher than that in normal guinea pigs fibroblasts.(3)Extraction of total protein from guinea pig iPS cells, western blot results show its high expression of two types of ES cell marker protein:oct4 and nanog. The results indicates that the guinea pig iPS cells are pluripotent.