316 INDUCTION OF PIG INDUCED PLURIPOTENT STEM CELLS BY RECOMBINANT PROTEINS ENCODED BY DEFINED FACTORS

Abstract

Pluripotent cells derived from any differentiated cell type through ectopic expression of transcription factors were designated as induced pluripotent stem (iPS) cells, exhibiting similar morphology and growth properties to embryonic stem (ES) cells besides expressing ES cell marker. Because iPS have the ability to differentiate into all types of cells, iPS cell technology is thought to have enormous potential for generating disease models, drug screening, toxicology, and regenerative medicine. However, for virus-mediated transfection of defined factors, the exogenous genes generally would be randomly inserted into the target cell’s genome, possibly bringing the potential hazard of insertional mutagenesis. Therefore, it is necessary to seek some new methods to induce somatic cell reprogramming without viruses. With instruction from the work of Zhou et al. (2009 Cell Stem Cell 4, 381–384), in the present study we attempted to use defined factors recombinant proteins-carried cell-penetrating peptide for the generation of porcine iPS cells, which would be a benefit for safe applications of iPS cells. Defined factors genes were amplified by PCR with specific primers of 9 arginines (R9) from recombinant plasmid pLL-hOCT4/pSox2/pMyc/pKlf4-EGFP (Yin et al. 2010 Prog. Biochem. Biophys. 37, 607–612) and inserted into prokaryotic expression vector pET-28a-EGFP. After DNA sequencing confirmation, the 4 recombinant plasmids were then transformed into BL21 strains, respectively. After IPTG induction, hOCT4/pSox2/pMyc/pKlf4-R9-EGFP fusion proteins were purified using Novagen His-Bind kit and confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, respectively. Then, defined factors recombinant proteins were added into pig fetal fibroblasts (PFF) medium every 48 h to establish pig iPS cells. The results showed that purified hOCT4/pSox2/pMyc/pKlf4-R9-EGFP fusion proteins could enter into PFF efficiently, and most of them were located in nuclei. The PPF were subcultured in stem cell medium condition and treated with defined factors recombinant proteins for 6 cycles simultaneously; the clear-cut cell colonies were gradually derived. These cells had large translucent nuclei and a high nucleo:cytoplasmic ratio and were positive for AP, Oct4, and Nanog. Detailed characterisation of such induced cells is ongoing. This research would provide new ideas for the induction of porcine somatic cell reprogramming.