| Animal somatic cells can be reprogrammed to induced pluripotent stem cells (iPS) that has recently become a hot research area. iPS cells have been generated from human, monkey, rat and pig, indicating that this technology could be widely used in establishing animal iPS cell lines. In this study, the goat embryonic fibroblasts (GEF) that was isolated from Guanzhong milk goat fetus were induced to reprogrammed cells (iPS) by four transcription factors in vitro. Based on Real-time RT-PCR method, we determined the expression of TERT in different tissues of Guanzhong milk goat fetus, gost fibroblasts and pips. We also conferred the relationship between goat reprogrammed cell formation and telomerase gene expression level. The main conclusions are as follows:1. The goat embryonic fibroblasts (GEF) from skin of Guanzhong milk goat fetus were isolated by using both the tissue culture and enzyme digestion methods. The isolated GEF had the good proliferation capability and the normal karyotype (60 XY). We chose the early passage (2-5) GEF as the recipient cells for the iPS induction.2. The relative expression level of eight organizations'telomerase (TERT) gene in Guanzhong dairy goat embryo was determined by using Real-time RT-PCR; and there had also varying degrees of TERT gene expression in other organizations. The results showed that the expression of TERT in testis was significantly higher than that of the epithelial tissue (p <0.01).3. Goat somatic cell reprogramming is a slow process, it normally takes more than 15-20d. Induced cells began to become a clone in 10-12d,and the cells around gradually began to gather,and finally formed a round colony which followed with a fibroblast-like shape to a round shape.The initial colony was small,and it can be continuously cultured after picking up the monoclonal, and it also can maintain the basic morphology.After 15d continuous culturation, the clone grew bigger, into a round or oval shape,and the edge of which is neat, the karyoplasmic ratio is relatively high, and then pick clones again and culture them continuously.4. AP stain positive clones in goat induced reprogramming cells were rarely. AP-positive clones were also positive in Oct4/Nanog/Tert immunofluorescence and Tert immunohistochemical staining, indicating that the reprogramming cells expressed endogenous gene and high telomerase activity.5. Using Real-time RT-PCR method to test the expression of TERT between reprogrammed primary cells and four incomplete reprogrammed cell lines, the results showed that the telomerase expression in alkaline phosphatase(AP)-positive reprogrammed cells was significantly higher than in AP-negative cells (p <0.01). |
