http://www.abbs.info E-mail: [email protected]

ISSN 1672-9145 Acta Biochim Biophys Sin 2005, 37(1): 68–73 CN 31-1940/Q

---

Establishment of Human Embryonic Stem Cell Line Stably Expressing Epstein-Barr Virus-Encoded Nuclear Antigen 1

Cai-Ping REN*, Ming ZHAO, Wen-Jiao SHAN, Xu-Yu YANG, Zhi-Hua YIN, Xing-Jun JIANG¹, Hong-Bo ZHANG, and Kai-Tai YAO

Cancer Research Institute, Central South University, Changsha 410078, China;

¹Neurosurgery Department, Xiangya Hospital, Central South University, Changsha 410008, China

Abstract Human embryonic stem (hES) cells have the capability of unlimited undifferentiated proliferation, yet maintain the potential to form perhaps any cell type in the body. Based on the high efficiency of the Epstein-Barr virus-based episomal vector in introducing exogenous genes of interest into mammalian cells, we applied this system to hES cells, expecting that this would resolve the problem of poor transfection efficiency existing in current hES cell research. Therefore, the first step was to establish EBNA1-positive hES cells. Using the Fugene 6 transfection reagent, we transfected hES cells with the EBNA1 expression vector and subsequently generated hES cell clones that stably expressed EBNA1 under drug selection. These clones were confirmed to express EBNA1 mRNA by RT-PCR and to express EBNA1 protein by Western blotting. Furthermore, luciferase reporter gene analysis was performed on the EBNA1 clones and revealed that the expressed EBNA1 protein was functional. When the EBNA1-positive cells were injected into severe combined immunodeficient (SCID) mice, they formed teratoma tissues containing all three embryonic germ layers and EBNA1 protein was detected in these teratoma tissues by Western blotting. All the results show that we have successfully created stable EBNA1-hES cells, thus laying a good foundation for further research.

Key words human embryonic stem cell; transfection; Epstein-Barr virus (EBV); EBNA1

-----------------

Received: September 9, 2004 Accepted: November 30, 2004

This work was supported by a grant from the National Natural Science Foundation of China (No. 30200140)

*Corresponding author: Tel, 86-731-2355066/4805451; Fax, 86-731-4360094; E-mail, [email protected]