Induction Of Pluripotent Stem Cells From Mouse Somatic Cell By Sheep Pluripotent Factors

Since the method of utilizing genetically modified sheep induced pluripotent stem cells (siPSCs) to acquire transgenic sheep has great prospect in the fields of agricultural industry and biomedicine, the study of sheep iPS has significant value. However it is still difficult to get fully reprogrammed sheep iPS cells effectively utilizing the existing sheep iPS technology. Therefore, the research of establishing thorough sheep iPS method receives much concern. Previously researchers mainly use mouse or human derived inducible factors to induce sheep pluripotent stem cells, but no reports have yet been seen about using sheep’s own factors. We found that sheep four factors (Oct4, Sox2, Klf4 and c-Myc, OSKM) have some difference with mouse or human factors in protein sequence by homology comparison, so the use of the heterogeneous factors may be one of the important reasons for the low efficiency and incompletely reprogramming of sheep iPS. In order to finally establish a thorough sheep iPS methods, this study built the sheep four pluripotency related genes (OSKM) cloned in preliminary work into the retroviral vector, and successfully induced mouse embryonic fibroblasts (MEF) into mouse iPS cells by viral packaging and infection, thus verified the induction effectiveness of sheep four genes. The results show that mouse iPS clones obtained in this study have typical mouse ES clone-like morphology; alkaline phosphatase (AP) tested positive; immunofluorescence assay showed the expression of three key pluripotency genes(Oct4, Nanog and Sox2); real-time quantitative PCR (qPCR) detection showed the transcriptional activation of endogenous ES cell marker genes (Oct4, ERas, Fgf4 and Zfp296); western blotting (WB) analysis showed iPSCs express OCT4 and SOX2 proteins. These results suggest that sheep factors-induced mouse iPSCs have similar morphology and molecular characteristics whit mouse ES cells, which showed that the four sheep factors built in this study have complete biological function and the ability of somatic cell reprogramming. In addition, by comparing with mouse four factors retroviral expression systems, this study showed that the sheep four factors retroviral expression systems have higher induction efficiency in mouse iPS. In conclusion, this research provides the basis of further using sheep’s own transcription factors to induce the sheep somatic cells into iPSCs.