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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
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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]