G Li1, Y Chunxu, L Guisheng. 1. Department of Oncology, the Fourth Affiliated Hospital, Medical College of Guangxi, 1 Liushi Road, Liuzhou, Guangxi, P. R. China, 545005. ligaofeng2012@sina.com
Abstract
OBJECTIVES: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.
OBJECTIVES: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.
Authors: Mia Buehr; Stephen Meek; Kate Blair; Jian Yang; Janice Ure; Jose Silva; Renee McLay; John Hall; Qi-Long Ying; Austin Smith Journal: Cell Date: 2008-12-26 Impact factor: 41.582