Abstract: The present study is to construct a eukaryotic expression vector of bovine Nanog, transfecting it into skin fibroblast, and further to obtain stable Nanog- transfected cell line, which can be used in nuclear transfer. By means of reverse transcription polymerase chain reaction (RT-PCR), cDNA of Nanog was cloned from tissues of fetal bovine primodial genital ridges at the age of about six weeks. It was inserted into pMD18-T vector, then into vector pCDNA3-FLAG by gene recombination technique. After being confirmed by restriction enzyme digestion and sequencing, the recombinant plasmid FLAG-Nanog was transfected into skin fibroblast cells. Selecting for two months with neomycine (G418), we obtained a stably transfected cell line. RT-PCR and Western Blotting were respectively used to detect the expression of Nanog mRNA and that of FLAG-Nanog fusion protein. In addition, immunostaining assay was performed to investigate whether the cell line is characteristic with stem cells. The results showed that (1) complete cording sequences (CDS) of Nanog was cloned from fetal bovine primodial genital ridges; (2) its eukaryotic expression vector was constructed, which expressed efficiently in the skin fibroblast cells; (3)Stage Specific Embryonic Antigen 4 (SSEA-4), factor Oct-4 as well as Nanog, required to maintain pluripotency of ES cells, was readily detectable in these Nanog-expressing fibroblast cells, indicating that the cell line is to some extent pluripotent. Our present study will lay a good foundation for further studying functions of Nanog, especially its role on early embryo development, production of transgenic animals, and derivation of embryonic stem cell lines from farm animals.