Department of Oncology, the Fourth Affiliated Hospital, Medical College of Guangxi, 1 Liushi Road, Liuzhou, Guangxi, P. R. China, 545005.
Cell Prolif. 2013 Feb;46(1):1-9. doi: 10.1111/cpr.12000. Epub 2012 Dec 21.
To generate rat induced pluripotent stem cells (iPSCs) using the PiggyBac (PB) transposon system, and to explore whether these iPSCs would be amenable to genetic manipulation.
The PB transposon system was used to reprogramme rat embryonic fibroblasts (EF) to become iPSCs. Cells were identified with regard to pluripotency and differentiation capacity in vitro and in vivo, population growth characteristics and gene expression; furthermore, targeting vector was electroporated into them. Correct recombination colonies were acquired by positive and negative selection, and then phenotype confirmed by Southern blotting.
The rat EF cells were reprogrammed into iPSCs successfully, using the PB transposon system. Cell morphology was found to display characteristics of rat embryonic stem cells (ESCs) and results of immunofluorescence staining and PCR indicated that they expressed pluripotency markers. In vivo and in vitro differentiation experiments proved that the cells could differentiate into all phenotypes from three germ layers, and to form chimaeras with high rat iPSC contribution. After electroporation with p53 targeting vector, approximately (5.44 ± 0.74) × 10(-6) colonies tolerated selection. Southern blotting confirmed that p53 gene was targeted successfully in the colonies.
The PB transposon system proved to be an effective method for reprogramming of rat EF cells into iPSCs. The rat iPSCs were amenable to gene targeting mediated by routine homologous recombination.
利用 PiggyBac（PB）转座子系统生成大鼠诱导多能干细胞（iPSCs），并探讨这些 iPSCs 是否易于进行基因操作。
利用 PB 转座子系统将大鼠胚胎成纤维细胞（EF）重编程为 iPSCs。通过体外和体内多能性和分化能力、群体生长特性和基因表达来鉴定细胞；此外，还将靶向载体电穿孔到细胞中。通过正负选择获得正确的重组菌落，然后通过 Southern 印迹进行表型确认。
利用 PB 转座子系统成功将大鼠 EF 细胞重编程为 iPSCs。细胞形态显示出大鼠胚胎干细胞（ESCs）的特征，免疫荧光染色和 PCR 结果表明它们表达多能性标记物。体内和体外分化实验证明，细胞可以分化为三个胚层的所有表型，并形成具有高大鼠 iPSC 贡献的嵌合体。用 p53 靶向载体电穿孔后，约有（5.44 ± 0.74）×10(-6)个菌落耐受选择。Southern 印迹证实了 p53 基因在菌落中成功靶向。
PB 转座子系统被证明是将大鼠 EF 细胞重编程为 iPSCs 的有效方法。大鼠 iPSCs 易于进行常规同源重组介导的基因靶向。
