Open AccessEfficient p53 gene targeting by homologous recombination in rat‐induced pluripotent stem cells
Author(s) -
Li G.,
Chunxu Y.,
Guisheng L.
Publication year - 2013
Publication title -
cell proliferation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.647
H-Index - 74
eISSN - 1365-2184
pISSN - 0960-7722
DOI - 10.1111/cpr.12000
Subject(s) - induced pluripotent stem cell , biology , reprogramming , embryonic stem cell , homologous recombination , electroporation , microbiology and biotechnology , gene targeting , genetics , gene
Objectives To generate rat induced pluripotent stem cells ( iPSC s) using the PiggyBac (PB) transposon system, and to explore whether these iPSC s would be amenable to genetic manipulation. Materials and methods The PB transposon system was used to reprogramme rat embryonic fibroblasts (EF) to become iPSC s. 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 iPSC s 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 iPSC s. The rat iPSC s were amenable to gene targeting mediated by routine homologous recombination.