Quantitative Analysis of Tert Expression in Goat Reprogramming Cells
|School||Northwest University of Science and Technology|
|Keywords||Telomerase Cell reprogramming Real-time RT-PCR Guanzhong dairy goat|
Animal somatic cells reprogrammed as induced pluripotent stem cells (iPS) stem cell biology research hotspot. In human, monkey, rat, pig, iPS cells have been described iPS technology can be widely used to create animal iPS cell lines. Guanzhong dairy goat fetus skin isolated fetal fibroblasts (GEF), to get the goat of reprogramming cells induced in vitro by four transcription factors. And induced goat reprogrammed cells and conducted a series of identification and detection. Real-time RT-PCR method Guanzhong dairy goat fetus organization TERT expression were detected, and then focus on goat somatic cell reprogramming process telomerase (TERT) relative gene expression levels were detected, explore the relationship goats re-programming cells in the formation of the telomerase gene expression. Concluded the following: 1. Taken Guanzhong dairy goat fetus skin, using tissue explant and trypsin digestion has been isolated and cultured fetal fibroblast cells (GEF). Proliferation ability, vigorous growth, normal karyotype (60 XY). GEF As the receptor induced goat iPS cells, can be seen by the growth curve of the number of passages as little as possible. Therefore, the selection of the first three generations of the GEF cells in experimental. By Real-time RT-PCR method for determination of Guanzhong dairy goat fetus eight kinds of organizations telomerase (TERT) the relative expression levels of the genes, TERT gene organization there are different degrees of expression. The results showed that the testicular tissue of TERT expression was significantly higher than that of other organizations (P lt; 0.01). 3 induced goat reprogramming somatic cells is a slow process, and generally require more than 15-20 d of culture time. Induced cells began to appear in the first 10-12 d clone colony surrounding cells gradually began to agglomerate into a circular shape and gathered into a colony from the bobbin-like morphology fibroblasts long. Smaller initial clone colony pick monoclonal can continue after subculturing, and to maintain the basic cell morphology. Sustain the culture 15d clone set off the grew up into a round or oval-shaped, neat edge, high quality nuclear picked clones were subcultured. 4. Induced goat reprogrammed cells the AP staining positive clones rarely. AP positive clone of Oct4, Nanog, TERT - immunofluorescence positive, Tert immunohistochemistry positive. Description the reprogrammed cells expression of the endogenous gene, expression of telomerase. 5 Real-time RT-PCR method reprogrammed cells of primary and four the incomplete reprogramming cell lines TERT expression test results found that the reprogramming of telomerase expression levels of alkaline phosphatase (AP) positive significantly higher than AP negative reprogrammed cells (P LT; 0.01). This revealed that the activation of telomerase activity and to maintain a higher expression level of reprogramming of somatic cells crucial.