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EMBRYONIC STEM CELLS

Establishment and Applications of Epstein-Barr Virus-Based Episomal Vectors in Human Embryonic Stem Cells

^a Cancer Research Institute, Xiang-Ya School of Medicine,
^c Department of Neurosurgery, Xiangya Hospital, Central South University, Changsha, Hunan, People’s Republic of China;
^b Regenerative Medicine Research Institute, Yunyang Medical College Tai-he Hospital, Shiyan, Hubei, People’s Republic of China

Key Words. Human embryonic stem cells • Episomal vectors • Epstein-Barr virus replicon • Stable gene transfer • Stable transgene expression • RNA interference

Correspondence: Caiping Ren, M.D., Cancer Research Institute, Xiang-Ya School of Medicine, Central South University, 110 Xiangya Road, Changsha 410078, Hunan, People’s Republic of China. Telephone: 86-731-2355066; Fax: 86-731-4360094; e-mail: rencaiping{at}xysm.net

Received July 27, 2005; accepted for publication December 27, 2005.
Human embryonic stem (hES) cells are capable of unlimited cell proliferation yet maintain the potential to differentiate into many cell types. Here we reported an Epstein-Barr virus (EBV)-based vector system used to improve transfection efficiency in hES cells. Plasmids containing oriP, the latent replication origin of EBV, can be propagated stably as episomal DNA in human cells that express the EBV nuclear antigen 1 (EBNA1), which binds to oriP and functions as the trans-acting replication initiator. It was reported that the EBV replicon could harbor a DNA fragment of up to 330 kilobase pairs. Plasmids containing an enhanced green fluorescent protein (EGFP)/puromycin resistance gene cassette along with or without oriP were used to transfect hES cells that stably express EBNA1. The presence of oriP moderately increased the transient transfection efficiency and more importantly it elevated the stable transfection efficiency by approximately 1,000-fold as compared with oriP-minus plasmids. The oriP plasmid as episomal DNA and green fluorescent protein expression in hES cells was maintained for months in the presence of drug selection and gradually lost (2%–4% per cell doubling) in the absence of selection. The presence of EBNA1 did not interfere with the hES cell properties or differentiation we tested and could maintain stable EGFP expression during differentiation. In addition to transgene expression, the EBV vector system could effectively enhance the RNA interference efficiency in hES cells. Thus, the EBV vector system that allows a large DNA insert and sustained expression of transgene or small hairpin RNA will enhance basic and translational research using hES cells.

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