| Induced pluripotent stem cells(iPSCs), very similar to embryonic stem cells(ESCs),can not only self-renewal but also holds the potential to differentiate into almost any type of cells, having a wide application in medicine, animal husbandry and biological basic research. However, low efficiency of induction, uncertainty of safety, and the poor quality still limited rapid development of iPSCs. The pig is an farm animal of important economic value, and represents an ideal animal model. Although isolation, culture and identification of porcine ESCs have been well documented, no bona fide porcine ESCs is derived so far,and the optimum culture conditions, identification criteria remain to be improved. Being regarded as a supplement of ESCs, pig iPSCs have been successfully obtained using a variety of induction strategies, vector delivery system and source cells. The successful induction of pig iPSCs are hoped to be helpful to the development of pig ESCs.Unfortunately, the induction efficiency of swine iPSCs is low, and the quality is poor as evidenced by poor germ line transmission competence. Pig iPSCs faces great challenge in producing chimeric pigs and cloned pigs. As known, the random integration of exogenous gene(s), supoptimal culture conditions can compromise the induction efficiency and quality of iPSCs. Using Tet on/off lentiviral inducible system, the present study aimed to explore effects of Vitamin C and /or CHIR-99021 addition on somatic reprogramming in order to optimize induction system; moreover, in order to lay solid foundation for future induction of pig iPSCs using recombinant proteins, transgene vectors harboring pluripotent transcription factors Oct4, Sox2, Klf4, c-Myc and Lin28 associated with permeable peptides were constructed followed by harvesting transgenic cell lines.Experiment Ⅰ: Optimization of pig somatic reprogramming of different source cells by addition of Vc and/or CHIR-99021 during induction.With porcine adipose stem cells, adult ear skin fibroblast cells, newborn piglets ear skin fibroblast cells and fetal fibroblast cells as the source cells, we utilized Tet-On lentiviral inducible system, which carried transcription factors Oct4, Sox2, Klf4, c-Myc to generate iPSCs. CHIR-99021 and / or Vc were added in the induction, and the number of positive clones was calculated by AP(Alkaline) positive staining, and the reprogramming efficiency of porcine somatic cells was compared among groups. The results showed that the Vc and CHIR-99021 significantly increased the reprogramming efficiency(P<0.05) of different pig types. In addition, somatic cell reprogramming efficiency of different source cell donor age and different type was compared. The results show that the effects of donor age of source cells on the reprogramming efficiency is not obvious(P > 0.05), but different types of somatic cells have significant difference(P < 0.05). Gene expression profiles at different stages during the induction of porcine adipose stem cells confirmed the happening of reprogramming.Experiment Ⅱ: Construction of eukaryotic transgenic vectors expressing Oct4-9R,Sox2-9R, Klf4-9R, c-Myc-9R and Lin28-9R and the establishment of transgenic cell lines.Total RNA was extracted from pig parthenogenetic blastocysts, iPSCs and adipose stem cells followed by reverse transcription into cDNA. PCR was performed to amplify coding region of pig Oct4, Sox2, Klf4, c-Myc and Lin28. pEGFP-N1 vector was used as backbone to construct the transgenic vectors by incorporating above metioned transcription factors into multiple cloning site. Gel electrophresis and sequencing results were employed to confirm that vectors were correctly constructed. We then used Fu GENE?HD to transfer vectors into 293 T cells. Puromycin was used to screen positively transfected 293 T for days.Transgenic 293 T cells with stable expression of Oct4-9R, Sox2-9R, Klf4-9R, c-Myc-9R,Lin28-9R and Ds RED-9R were obtained. To examine membrane permeability of recombinant proteins, pig ear fibroblasts and mouse embryonic fibroblast were incubated with the Ds RED-9R protein, purified from 293 T cells, for 8h. The results demonstrated that Ds RED-9R can transfer into the cytoplasm and nucleus, which proved that the 9R is functional to carry the associated protein into the cytoplasm and nucleus.In conclusion, VC and/or CHIR-99021 of appropriate concentration and treatment duration can improve somatic reprogramming efficiency of different source cells in pig;eukaryotic transgenic vectors harboring Oct4-9R, Sox2-9R, Klf4-9R, c-Myc-9R and Lin28-9R associated with cell penetrating peptide were successfully constructed, and transgenic seed cells expressing defined factors were also obtained. These results lay a foundation for obtainning the non integrated genome of porcine iPSCs in the future. |
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