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Preliminary Study on Nucleofection of Rat Embryonic Stem Cells
Wu Zhaoqiang, Zhao Lihua, Zhang Manling, Jin Yong, Nie Xiaowei, Hou Daorong, Yang Ning, Chen Yuan, Li Rongfeng*
Key Laboratory of Xenotransplantation of Jiangsu Province, Nanjing Medical University, Nanjing 210029, China
Abstract: The study of gene transduction on rat embryonic stem cells (rESCs) has been explored continually. In our study， in order to detect the optimized program for gene transduction， the reporter red fluorescent protein (RFP) gene was transfected into rESCs with different nucleofection programs. The proportion of cells with red fluorescent and the dead cells after transfection were compared among different programs. Then， the best program was selected and used to transfect rESCs. Alkaline phosphatase (AP) staining， RT-PCR and immunostaining were performed to compare the characters of rESCs before and after nucleofection. The results showed that the A-13 was the best program for rESCs transfection and its efficiency reached (39.63±1.75)%. The AP staining of rESCs was still positive after nucleofection， and the expression and immunostaining of ESC marker genes (RT-PCR and immunostaining) were not changed by the nucleofection process. Therefore， the rESCs could be effectively transducted with the nucleofection program A-13 and their morphology and pluripotency characters were maintained. Nucleofection was an effective method for gene transduction of rESCs.
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