825 STEM CELLS AND DEVELOPMENT 17:825–836 (2008) © Mary Ann Liebert, Inc. DOI: 10.1089/scd.2007.0233 Introduction H uman embryonic stem cells (hESC) are pluripotent cells derived from the inner cell mass of the blastocyst. They are characterized by their ability to proliferate indefinitely in culture as undifferentiated cells with a normal karyotype and have the potential to differentiate into various cell types. Therefore, the cellular therapy applications of hESC rely on the efficient genetic modification of the hESC in vitro. The techni- cal development of gene transfer into hESC has recently been reviewed [1–4]. A few studies have described the transfec- tion of hESC for generation of lines stably expressing specific transgenes with endogenous or viral promoters [4–10], but the best method that gives reliable results for routinely produc- ing stable transfectants is still uncertain. Recently, although lentiviral transduction was proven to produce a high propor- tion of stable integrants in hESC [11–14], its application can be limited by vector size and time-consuming efforts to con- struct vectors for lentivirus production. However, despite all these reports, the optimal choice of promoter system for driv- ing transgene expression in hESC is greatly elusive. Various cellular and viral promoters have been used for genetic modi- fication of cells by exogenous transgene expression [15–17]. One of the most popular choices has been the cytomegalo- virus (CMV) promoter because of its strong activity in most Generation of High-Level Stable Transgene Expressing Human Embryonic Stem Cell Lines Using Chinese Hamster Elongation Factor-1  Promoter System Ken Kwok-Keung Chan, 1 Selena Meiyun Wu, 1 Peter Morin Nissom, 2 Steve K.W. Oh, 1 and Andre B.H. Choo 1 The utilization of human embryonic stem cells (hESC) in regenerative medicine largely depends on the development of technologies that will allow efficient genetic manipulation of the cells in vitro. Although a few studies have described the transfection of hESC for generation of reporter lines stably expressing specific trans- genes driven by different promoters, the optimal choice of promoter system for driving transgene in hESC has yet to be elucidated. We show for the first time that Chinese hamster elongation factor-1 α (CHEF1) promoter robustly drove reporter gene expression higher than the human elongation factor 1 α (hEF1 α ), other constitutive Chinese hamster promoters, human cytomegalovirus (CMV) immediate early enhancer/promoter and SV40 pro- moters in hESC by quantitative analysis. We also successfully generated stably transfected hESC lines using this CHEF1 promoter system and demonstrated that they continued to express enhanced green fluorescent protein (EGFP) during prolonged undifferentiated proliferation, in differentiated embryoid bodies (EBs), and in terato- mas without transgene silencing. By immunofluorescence staining and flow cytometry analysis, the pluripotent markers, OCT-4, SSEA-4, and TRA-1-60, continued to be expressed in undifferentiated CHEF1-EGFP expressing hESC lines. When the stably transfected hESC were directed to differentiate into neural precursors in vitro, high-level EGFP expression was maintained and co-expression of neural markers, Nestin, and β-tubulin III was observed. The morphology, karyotype, and telomerase activity of CHEF1-EGFP expressing hESC were normal after >50 continuous passages, and the cells retained the ability to differentiate into derivatives of the three germ layers in vitro as confirmed by RT-PCR analysis and immunocytochemical staining and in vivo teratoma formation. Therefore, stable CHEF1-EGFP hESC lines retained the capability for self-renewal and pluripotency. The novel CHEF1 promoter system described here enables high-level transgene expression in the stably trans- fected hESC. It may have significant implication for uses in bioprocess development and future development of gene-modified hESC in tissue regeneration and transplantation applications. 1 Stem Cell Group and 2 Microarray Laboratory, Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), Biopolis, Singapore. Downloaded by 54.162.69.248 from www.liebertpub.com at 05/31/20. For personal use only.