Past Issue

Volume 19, Number 4, Jan-Mar(Winter) 2018, Serial Number: 76, Pages: 640-646

Effects of Klf4 and c-Myc Knockdown on Pluripotency Maintenance in Porcine Induced Pluripotent Stem Cell

Yu-Jing Liao, M.Sc, 1, 2, Yi-Shiou Chen, B.Sc, 1, Ja-Xin Lee, B.Sc, 3, Lih-Ren Chen, Ph.D, 1, 4, 5, Jenn-Rong Yang, Ph.D., 1, *,
Division of Physiology, Livestock Research Institute, Council of Agriculture, Executive Yuan, Tainan, Taiwan
Department of Animal Science, National Chung Hsing University, Taichung, Taiwan
Hsinchu Branch, Livestock Research Institute, Council of Agriculture, Executive Yuan, Hsinchu, Taiwan
Institute of Biotechnology, National Cheng Kung University, Tainan, Taiwan
Institute of Biotechnology, Southern Taiwan University, Tainan, Taiwan
*Corresponding Address: Physiology Division Livestock Research Institute Council of Agriculture Executive Yuan Tainan Taiwan. 112 Farm Rd. Hsinhua Tainan 71246 Taiwan



The importance of Oct4 and Sox2 in maintaining pluripotency and self-renewal is well-understood, but the functions of Klf4 and c-Myc has not been fully investigated. In the present study, we attempted to determine the roles of Klf4 and c-Myc on pluripotency maintenance of porcine induced pluripotent stem (piPS) cells.

Materials and Methods

In this experimental study, we performed short hairpin RNA (shRNA) to knock down the Klf4 and c-Myc functions of piPS cells and examined pluripotency markers and teratoma formation to evaluate piPS cell pluripotency. The shRNA-Klf4 and shRNA-c-Myc vectors containing a reporter gene, TagFP635, were transfected into piPS cells by lentivirus infection. The piPS cells fully expressing infrared fluorescence were selected to confirm gene knockdown of Klf4 and c-Myc reverse transcription-polymerase chain reaction (RT-PCR). Next, for pluripotency evaluation, expression of pluripotency markers was detected by immunocytochemical staining, and capability of teratoma formation was investigated by piPS cell transplantation into nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice.


Our findings indicated that Klf4 and c-Myc functions of piPS cells were knocked down by shRNA transfection, and knockdown of Klf4 and c-Myc functions impaired expression of pluripotency markers such as Oct4, AP, SSEA-3, SSEA-4, TRA-1-6, and TRA-1-81. Furthermore, piPS cells without Klf4 and c-Myc expression failed to form teratomas.


The pluripotency of piPS cells are crucially dependent upon Klf4 and c-Myc expression. These findings, suggesting potential mechanisms of Klf4 and c-Myc contribution to piPS cell formation, have important implications for application, regulation, and tumorigenesis of piPS cells.