The Study Of Reprogramming Goat Ear Fibroblasts Into Induced Of Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) were found by Yamanaka and Yamanaka from the university of Tokyo in2006. As iPSCs are very similar to the embryonic stem cells(ES) in self-renew and differentiation potential and without involving in ethical issues, at the same time, they have great application potential in clinical reserarh and have become the focus of current research quickly.On an attempt to generate goat induced pluripotent stem cells in this study, goat ear fibroblasts were isolated from adult goat and reprogram goat ear fibroblasts into goat induced pluripotent stem cells with a polycistronic plasmid delivery system and inducible lentiviruses system. The main findings were as follows:1. Goat ear fibroblasts isolated from goat ear and mouse embryonic fibroblasts isolated from C57/BL6mice, they were treated with mitomycin C to be used as feeder.3. Lentiviral vectors and packeging system were cotransfected into293t cells, after48h, green fluorescent protein can be visible under a fluosrescence microscope, and then the lentivirus supernatant was collected and titered. The titers of lentivirus plentG-KOSM and drug-inducible lentivirus reached105~6TU/ml and106-7TU/ml respectively. Then make the titers of lentivirus plentG-KOSM reached106-7TU/ml by Millipore100KD concentrating column.4. Two sets of lentivirus were transduced into goat ear fibroblasts and cultured with different feeders. Results showed that we only got suspected induced pluripotent stem cells with mouse embryonic fibroblasts feeder by drug-inducible lentivirus.5. Identification of biological characteristics of goat iPS cells:1) The goat iPS cells were positive with alkaline phosphatase staining, and immunofluorescence staining demonstrated that goat iPS cells expressed ES cell maker protein Tra-1-60, Tra-1-81, REX-1and SSEA-1.2) The RNA of suspected induced pluripotent stem cells was isolated and RT-PCR results showed that they expressed embryonic stem cell marker genes such as Rexl、. CDH1、Dnmt3b、TDGF、Sox2and Oct4. 3) DNA demethylation analysis the promoters of nanog gene revealed that, the promoters of nanog was hypo methylated in goat induced pluripotent stem cells and hypermethylated in goat ear fibroblasts.4) Suspected goat induced pluripotent stem cells could form embryoid bodies(EBs) in vitro, and EBs could differentiate into various cells.They express3-germ layers marker genes such as Fibronectin(ectoderm)、Enolase3(mesoderm)、AFP(entoderm).5) Approximately5×106of goat induced pluripotent stem cells were inject into NOD/SCID mice, and teratoma was formatted.These results showed thar goat induced pluripotent stem cells had potential in vivo.