Studies On The Promotion Of Porcine Somatic Cell Reprogramming By Embryonic Stem Cell-specific MicroRNAs

The differentiated state of a somatic nucleus can be reversed to an undifferentiated stem cell with pluripotent state, which is defined as somatic reprogramming. Reprogramming would be useful for biomedical research and transgenic breeding. Pig is the most important economic animal in china, the research of porcine somatic cells reprogramming molecular mechanisms will promote the development of biomedical and agricultural. Somatic cell nuclear transfer (SCNT) technique offer tremendous promise for future development of patient-specific therapy. But the efficiency of reprogramming is still low, and the experimental operation is complicated and time-consuming. Induced pluripotent stem cells (iPS) technique, have been demonstrated could reprogram murine and human somatic cells to pluripotent state by defined transcription factors, without destroying embryos. This study research on the porcine somatic cells reprogramming and the promotion of the reprogramming efficiency by embryonic stem cell-specific microRNAs (miRNAs) using SCNT technique and iPS technique.Firstly, we successfully cloned a Chinese Taihu pig by SCNT using fetal fibroblasts of Taihu pigs. The analysis of microsatellite genotyping for Taihu Pigs confirmed that the piglet was genetically derived from fetal fibroblasts of Taihu pigs and unrelated to their surrogate mother. The birth of the first cloned Taihu pig lays the foundation for generation of genetically modified Taihu pigs with high productivity or for human disease models.Secondly, we transfected 4 transcription factors (Oct4,Klf4,Sox2.c-Myc) into porcine somatic cells using retroviral system. We obtained iPS colonies which exhibited typical undifferentiated human ES-cell-like morphology. These iPS colonies displayed high nuclear/cytoplasmic ratio, and were AP positive. Semiquantitative Reverse-transcription PCR (RT-PCR) demonstrated integration of the four transgenes into the genome of all tested iPS cell lines, and expression of endogenous Oct4, Nanog, Lin28 and Sox2 robustly increased. Indicative of acquisition of pluripotency characteristics:porcine iPS colonies stained positive for SSEA4,Oct4, Nanog and Rexl (Zfp42). Furthermore, cells were able to form embryoid bodies after 3 days hanging drop culture. RT-PCR analysis confirmed the expression of AFP, alphaAT, BMP4, Enolase, GFAP and Neurod genes, which are the marker genes of three germ layers. In vivo testing of iPS cell lines for their developmental potential (teratoma and chimera assay) is currently underway.We supposed that pluripotency relevant miRNAs could improve the reprogramming efficiency of porcine somatic cells. Here, we selected seven kinds of miRNAs including miR-302a, miR-302d, miR-371, miR-372, miR-200c, miR-294, and miR-17, which were introduced with retroviruses expressing Oct4, Sox2, Klf4 and C-myc into porcine embryonic fibroblasts (PEF). MiR-302a & miR-302d and miR-200c mimics showed the greatest effects. When they transfected with 4 retrovirus factors, the efficiency was increased from 0.01-0.05% to 0.2-0.3% of transduced PEFs, and the induced days were reduced from 12 to 10. When they transfected with 3 retrovirus factors, the efficiency was increased from 0.001-0.005% to 0.01-0.05% of transduced PEFs, and the induced days were reduced from 14to 12. Our data show that miRNAs can boost the reprogram of porcine somatic cells into iPS cells. This study provides a reference for generation porcine iPS cells and the research of reprogramming molecular mechanisms.