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
Livestock Research Institute
Council of Agriculture
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Effects of Klf4 and c-Myc Knockdown on Pluripotency Maintenance
in Porcine Induced Pluripotent Stem Cell .
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
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.