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Nanog is a homeodomain transcription factor that is expressed specifically in undifferentiated embryonic stem (ES) cells and has been shown to be essential in the maintenance of pluripotency in mouse ES cells. To examine the function of NANOG in primate ES cells, we generated transgenic monkey ES cell lines expressing three- to seven-fold higher levels of NANOG protein compared to wild-type ES cells. These NANOG over-expressing cell lines retained their undifferentiated state in the absence of a feeder layer, as shown by expression of undifferentiated ES cell markers such as alkaline phosphatase (ALP) and OCT-4. We also demonstrated that in vitro differentiation of transgenic cell lines was mostly restricted to the ectodermal lineage, as examined by reverse transcriptase-polymerase chain reaction (RT-PCR). Knockdown experiments using NANOG small interfering (si) RNA resulted in induction of differentiation markers such as AFP, GATA4 and GATA6 for the endoderm and CDX2 for the trophectoderm. These results suggest that NANOG plays a crucial role in maintaining the pluripotent state of primate ES cells.