The Research On Production And Appraisal Of Sheepinduced Pluripotent Stem Cells

In 2006, The recombinant plasmid containing Oct4, Sox2, Klf4, and c-Myc gene respectively was transfected into the fibroblasts cell of mice by viral vector by Yamanaka, Induced pluripotent stem cells(i PS cells) they obtaind, which were like embryonic stem cells,provided a new direction for cell research, also brought the broad prospect of application in regenerative medicine field. Animal i PS cells have a great influence on disease model, cell therapy, the preservation of germplasm resources, and the raise of the production efficiency of somatic cell cloned animals, etc.objective : Under the condition of feederfree to establish the preparation methods of i PS cells by electroporation transfection of five factors, the technology of the i PS cells induced lay the foundation of further study the cell reprogramming mechanisms and the future clinical application of i PS cells.Methods:(1)This experiment isolated Chinese merino fibroblasts by tissue culture method, the double enzymatic method, and subculture.(2) three recombinant plasmids containing the human gene of Oct4, Sox2 and Klf4, c-Myc and lin28, respectively, was transfected into the sheep fibroblasts cell by 4-D electroporation. The suitable procedure for sheep fibroblasts cell was screened from EH-100, EN-150, CZ-167, CA-137. The impact on i PS cell colony formation was studied in different conditions for choosing the most suitable for the preparation of i PS, such as: Cell density and plasmid concentration are consistent, different plasmid proportion impact on the positive AP staining(AP +) colony rate; plasmid proportion and the plasmid concentration are consistent, Cell density impact on the positive AP staining(AP+) colony rate; Cell density and plasmid proportion are consistent, the plasmid concentration impact on the positive AP staining(AP+) colony rate; Cell density, plasmid concentration, plasmid proportion are consistent, medium, with or without adding Vc, the impact on the positive AP staining(AP+) colony rate.(3)Through appearance observation and quantitative PCR, immunofluorescence staining, transcriptome sequencing to detect sheep class i PS cells.Results and conclusion:(1)Double enzymatic rapid success isolated source of Chinese merino sheep ear fibroblasts, oriented programming provides excellent precursors.(2)The results indicated that the EH-100, the cell concentration of 5×106cell / m L; plasmid ratio of 1: 1: 1, plasmid concentration of 0.1 mg/L and the culture with 0.05 mg/L Vc were the suitable program, and the positive AP staining(AP +) colony rate reaching 14% was hinger than any other progamme.(3)Under the condition of feederfree, five factors will import source of sheep ear fibroblasts by electroporation transfection, and reprogrammed the class i PS cells successfully, shape, pluripotent gene expression, the transcriptome of appraisal results show that it has more potential, It laid a theory foundation for the further research of sheep mechanism of pluripotent stem cells.